U.S. patent application number 11/188287 was filed with the patent office on 2006-01-26 for compounds, compositions, and methods.
This patent application is currently assigned to Cytokinetics, Inc.. Invention is credited to Gustave Bergnes, Dashyant Dhanak, Steven David Knight, Pu-Ping Lu, Andrew McDonald, David J. JR. Morgans.
Application Number | 20060019988 11/188287 |
Document ID | / |
Family ID | 29740126 |
Filed Date | 2006-01-26 |
United States Patent
Application |
20060019988 |
Kind Code |
A1 |
McDonald; Andrew ; et
al. |
January 26, 2006 |
Compounds, compositions, and methods
Abstract
Compounds useful for treating cellular proliferative diseases
and disorders by modulating the activity of KSP are disclosed.
Inventors: |
McDonald; Andrew; (San
Francisco, CA) ; Lu; Pu-Ping; (Foster City, CA)
; Bergnes; Gustave; (Pacifica, CA) ; Morgans;
David J. JR.; (Los Altos, CA) ; Dhanak; Dashyant;
(West Chester, PA) ; Knight; Steven David; (West
Chester, PA) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Cytokinetics, Inc.
Smithkline Beecham Corporation
|
Family ID: |
29740126 |
Appl. No.: |
11/188287 |
Filed: |
July 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10462002 |
Jun 12, 2003 |
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11188287 |
Jul 21, 2005 |
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60389265 |
Jun 14, 2002 |
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60389779 |
Jun 18, 2002 |
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Current U.S.
Class: |
514/309 ;
546/141 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
29/00 20180101; A61P 37/00 20180101; A61P 35/00 20180101; A61P
43/00 20180101; C07D 401/06 20130101; C07D 217/24 20130101 |
Class at
Publication: |
514/309 ;
546/141 |
International
Class: |
C07D 217/22 20060101
C07D217/22; A61K 31/47 20060101 A61K031/47 |
Claims
1. A method of inhibiting KSP kinesin activity which comprises
contacting said kinesin with an effective amount of a compound
having the structure represented by Formula I: ##STR35## wherein:
R.sub.1 is chosen from optionally substituted
phenyl-C.sub.1-C.sub.4-alkyl-, optionally substituted
heteroaryl-C.sub.1-C.sub.4-alkyl-, and naphthalenylmethyl-; R.sub.2
and R.sub.2' are independently chosen from hydrogen, optionally
substituted alkyl-, optionally substituted alkoxy, optionally
substituted aryl-, optionally substituted aralkyl-, optionally
substituted heteroaryl-, and optionally substituted heteroaralkyl-;
or R.sub.2 and R.sub.2' taken together form an optionally
substituted 3- to 7-membered ring; R.sub.12 is selected from the
group consisting of optionally substituted imidazolyl-, optionally
substituted imidazolinyl-, --NHR.sub.4; --N(R.sub.4)(COR.sub.3);
--N(R.sub.4)(SO.sub.2R.sub.3a); and --N(R.sub.4)(CH.sub.2R.sub.3b);
R.sub.3 is chosen from hydrogen, optionally substituted alkyl-,
optionally substituted aryl-, optionally substituted aralkyl-,
optionally substituted heteroaryl-, optionally substituted
heteroaralkyl-, R.sub.15O-- and R.sub.17--NH--; R.sub.3a is chosen
from optionally substituted alkyl-, optionally substituted aryl-,
optionally substituted aralkyl-, optionally substituted
heteroaryl-, optionally substituted heteroaralkyl-, and
R.sub.17--NH--; R.sub.3b is chosen from hydrogen, optionally
substituted alkyl-, optionally substituted aryl-, optionally
substituted aralkyl-, optionally substituted heteroaryl-, and
optionally substituted heteroaralkyl-; R.sub.4 is chosen from
hydrogen, optionally substituted alkyl-, optionally substituted
aryl-, optionally substituted aralkyl-, optionally substituted
heterocyclyl-, and optionally substituted heteroaralkyl-; R.sub.5,
R.sub.6, R.sub.7 and R.sub.8 are independently chosen from
hydrogen, optionally substituted alkyl, optionally substituted
alkoxy, halogen, hydroxyl, nitro, cyano, dialkylamino,
alkylsulfonyl, alkylsulfonamido, alkylthio, carboxyalkyl,
carboxamido, aminocarbonyl, optionally substituted aryl and
optionally substituted heteroaryl; R.sub.15 is optionally
substituted alkyl-, optionally substituted aryl-, optionally
substituted aralkyl-, optionally substituted heteroaryl-, or
optionally substituted heteroaralkyl-; and R.sub.17 is chosen from
hydrogen, optionally substituted alkyl, optionally substituted
aryl, optionally substituted aralkyl, optionally substituted
heteroaryl, and optionally substituted heteroaralkyl; a
pharmaceutically acceptable salt of a compound of Formula I; a
pharmaceutically acceptable solvate of a compound of Formula I; or
a pharmaceutically acceptable solvate of a pharmaceutically
acceptable salt of a compound of Formula I.
2-5. (canceled)
6. A method according to claim 1, wherein R.sub.1 is benzyl.
7. A method according to claim 1, wherein R.sub.2 and R.sub.2' are
independently chosen from hydrogen, optionally substituted alkyl-,
optionally substituted alkoxy, optionally substituted aryl-,
optionally substituted aralkyl-, optionally substituted
heteroaryl-, and optionally substituted heteroaralkyl-; or R.sub.2
and R.sub.2' taken together form an optionally substituted 3- to
7-membered ring.
8. A method according to claim 7, wherein R.sub.2 is optionally
substituted C.sub.1-C.sub.4 alkyl-, and R.sub.2' is hydrogen or
optionally substituted C.sub.1-C.sub.4 alkyl-.
9. A method according to claim 8, wherein R.sub.2' is hydrogen and
R.sub.2 is optionally substituted C.sub.1-C.sub.4 alkyl-.
10. A method according to claim 9, wherein R.sub.2' is hydrogen and
R.sub.2 is ethyl or propyl.
11. A method according to claim 10, wherein R.sub.2 is
i-propyl.
12. A method according to claim 1, wherein if either R.sub.2 or
R.sub.2' is hydrogen, then the other is not hydrogen.
13. A method according to claim 1, wherein R.sub.5, R.sub.6,
R.sub.7, and R.sub.8 are independently chosen from hydrogen,
hydroxyl, halogen, optionally substituted C.sub.1-C.sub.4 alkyl-,
C.sub.1-C.sub.4 alkoxy, cyano, amino, substituted amino, or
carbamyl-.
14. A method according to claim 13, wherein R.sub.5, R.sub.6,
R.sub.7, and R.sub.8 are independently methoxy, methyl,
trifluoromethyl, cyano, hydrogen or halo.
15. A method according to claim 14, wherein only one of R.sub.5,
R.sub.6, R.sub.7, and R.sub.8 is not hydrogen.
16. A method according to claim 1, wherein R.sub.12 is an
optionally substituted imidazolyl- having the formula: ##STR36##
wherein R.sub.9 is chosen from hydrogen, optionally substituted
C.sub.1-C.sub.8 alkyl-, optionally substituted aryl-, optionally
substituted aryl-C.sub.1-C.sub.4-alkyl-, optionally substituted
heteroaryl-C.sub.1-C.sub.4-alkyl-, optionally substituted
aryl-C.sub.1-C.sub.4-alkoxy, optionally substituted
heteroaryl-C.sub.1-C.sub.4-alkoxy, and optionally substituted
heteroaryl-; and R.sub.13 and R.sub.13' are independently hydrogen,
optionally substituted C.sub.1-C.sub.8 alkyl-, optionally
substituted aryl-, or optionally substituted
aryl-C.sub.1-C.sub.4-alkyl-.
17. A method according to claim 16, wherein R.sub.9 is lower-alkyl;
phenyl-; or phenyl- substituted with one or more of the following
groups: methyl, methoxy, trifluoromethyl, or halo.
18. A method according to claim 16, wherein R.sub.13 is hydrogen
and R.sub.13' is optionally substituted substituted C.sub.1-C.sub.4
alkyl-.
19. A method according to claim 1, wherein R.sub.12 is an
optionally substituted imidazoline having the formula ##STR37##
wherein, R.sub.14 is chosen from hydrogen, optionally substituted
C.sub.1-C.sub.8 alkyl-, optionally substituted aryl-, optionally
substituted aryl-C.sub.1-C.sub.4-alkyl-, optionally substituted
heteroaryl-, optionally substituted
heteroaryl-C.sub.1-C.sub.4-alkyl-; and R.sub.10, R.sub.10',
R.sub.11 and R.sub.11' are independently chosen from hydrogen,
optionally substituted C.sub.1-C.sub.8 alkyl-, optionally
substituted aryl-, and optionally substituted
aryl-C.sub.1-C.sub.4-alkyl-.
20. A method according to claim 19, wherein R.sub.10, R.sub.10',
R.sub.11', and R.sub.11 are independently hydrogen or optionally
substituted C.sub.1-C.sub.4 alkyl-.
21. A method according to claim 1 wherein R.sub.12 is --NHR.sub.4,
--NR.sub.4(COR.sub.3), NR.sub.4(SO.sub.2R.sub.3a), or
--NR.sub.4(CH.sub.2R.sub.3b) and R.sub.4 is R.sub.16-alkylene-, and
R.sub.16 is chosen from alkoxy, amino, alkylamino, dialkylamino,
carboxy, guanidine, hydroxyl-, and N-heterocyclyl-.
22. A method according to claim 1 wherein R.sub.12 is
--NR.sub.4(COR.sub.3) and R.sub.3 is selected from hydrogen,
optionally substituted alkyl-, optionally substituted aralkyl-,
optionally substituted heteroaralkyl-, optionally substituted
heteroaryl-, optionally substituted aryl-, R.sub.15O-- and
R.sub.17--NH--, wherein R.sub.15 is chosen from optionally
substituted alkyl and optionally substituted aryl and R.sub.17 is
chosen from hydrogen, optionally substituted alkyl and optionally
substituted aryl-.
23. A method according to claim 22 wherein R.sub.12 is
--NR.sub.4(COR.sub.3) and R.sub.3 is tolyl-, halophenyl-,
halomethylphenyl-, hydroxymethylphenyl-, methylenedioxyphenyl-,
formylphenyl or cyanophenyl-.
24. A method according to claim 21 wherein R.sub.3a is chosen from
C.sub.1-C.sub.13 alkyl-; phenyl-; naphthyl-; phenyl substituted
with cyano, halo, lower-alkyl-, lower-alkoxy, nitro,
methylenedioxy, or trifluoromethyl-; biphenylyl and
heteroaryl-.
25. A method according to claim 21 wherein R.sub.3b is chosen from
C.sub.1-C.sub.13 alkyl-; substituted lower-alkyl-; phenyl-;
naphthyl-; phenyl substituted with cyano, halo, lower-alkyl-,
lower-alkoxy, nitro, methylenedioxy, or trifluoromethyl-;
biphenylyl-, benzyl and heterocyclyl-.
26. A method according to claim 1 wherein, R.sub.1 is optionally
substituted aryl-C.sub.1-C.sub.4-alkyl-, optionally substituted
heteroaryl-C.sub.1-C.sub.4-alkyl-, or naphthalenylmethyl R.sub.2 is
optionally substituted C.sub.1-C.sub.4-alkyl-; R.sub.2' is
hydrogen; R.sub.7 is hydrogen, methyl, or cyano; R.sub.5, R.sub.6,
and R.sub.8 are hydrogen; and R.sub.12 is optionally substituted
imidazolyl-, optionally substituted imidazolinyl-, --NHR.sub.4;
--N(R.sub.4)(COR.sub.3); --N(R.sub.4)(SO.sub.2 R.sub.3a); and
--N(R.sub.4)(CH.sub.2R.sub.3b).
27. A method according to claim 26 wherein R.sub.12 is optionally
substituted imidazolyl- having the formula: ##STR38## wherein
R.sub.9 is lower-alkyl; phenyl-; or phenyl- substituted with one or
more of the following groups: methyl, methoxy, trifluoromethyl, or
halo; R.sub.13 is hydrogen; and R.sub.13' is substituted
C.sub.1-C.sub.4 alkyl-.
28. A method according to claim 26 wherein R.sub.12 is optionally
substituted imidazolinyl- having the formula: ##STR39## wherein
R.sub.14 is lower-alkyl; phenyl-; or phenyl- substituted with one
or more of the following groups: methyl, methoxy, trifluoromethyl,
or halo; and R.sub.10, R.sub.10', R.sub.11', and R.sub.11 are
independently hydrogen or optionally substituted C.sub.1-C.sub.4
alkyl-.
29. A method according to claim 26 wherein R.sub.12 is --NHR.sub.4;
and R.sub.4 is chosen from hydrogen, optionally substituted alkyl-,
optionally substituted aryl-, optionally substituted aralkyl-,
optionally substituted heteroaralkyl-, and optionally substituted
heterocyclyl-.
30. A method according to claim 26 wherein R.sub.3 is selected from
hydrogen, optionally substituted alkyl-, optionally substituted
aralkyl-, optionally substituted heteroaralkyl-, optionally
substituted heteroaryl-, optionally substituted aryl-, R.sub.15O--
and R.sub.17--NH--, wherein R.sub.15 is chosen from optionally
substituted alkyl and optionally substituted aryl and R.sub.17 is
chosen from hydrogen, optionally substituted alkyl and optionally
substituted aryl.
31. A method according to claim 30 wherein R.sub.4 is is
R.sub.16-alkylene-, and R.sub.16 is chosen from alkoxy, amino,
alkylamino, dialkylamino, carboxy, hydroxyl-, and
N-heterocyclyl-.
32. A method according to claim 31, wherein R.sub.4 is chosen from
hydrogen, optionally substituted alkyl-, optionally substituted
aryl-, optionally substituted aralkyl-, optionally substituted
heteroaralkyl-, and optionally substituted heterocyclyl- and
R.sub.3 is selected from optionally substituted alkyl-; aryl-;
substituted aryl-; benzyl-; and optionally substituted
heteroaryl-.
33. A method according to claim 32, wherein R.sub.3 is tolyl-,
halophenyl-, halomethylphenyl-, hydroxymethylphenyl-,
methylenedioxyphenyl-, formylphenyl or cyanophenyl-.
34. A method according to claim 26, wherein R.sub.12 is
--N(R.sub.4)(CH.sub.2R.sub.3b); R.sub.4 is chosen from hydrogen,
optionally substituted alkyl-, optionally substituted aryl-,
optionally substituted aralkyl-, optionally substituted
heteroaralkyl-, and optionally substituted heterocyclyl- and
R.sub.3b is chosen from phenyl substituted with one or more halo,
methyl-, cyano, trifluoromethyl-, trifluoromethoxy, carboxy, or
methoxycarbonyl groups; piperidinyl-; and naphthyl-.
35. A method according to claim 1, wherein R.sub.12 is
--NR.sub.4(SO.sub.2R.sub.3a); R.sub.4 is chosen from hydrogen,
optionally substituted alkyl-, optionally substituted aryl-,
optionally substituted aralkyl-, optionally substituted
heteroaralkyl-, and optionally substituted heterocyclyl- and
R.sub.3a is chosen from phenyl substituted with halo, lower-alkyl-,
lower-alkoxy, cyano, nitro, methlenedixoy, or trifluoromethyl-; and
naphthyl-.
36. A method according to claim 1, wherein R.sub.2 and R.sub.2' are
each attached to a stereogenic center having an
R-configuration.
37-48. (canceled)
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/389,265, filed Jun. 14, 2002 and of U.S.
Provisional Patent Application No. 60/389,779, filed Jun. 18, 2002;
each of which is incorporated herein by reference for all
purposes.
FIELD OF THE INVENTION
[0002] This invention relates to compounds which are inhibitors of
the mitotic kinesin KSP and are useful in the treatment of cellular
proliferative diseases, for example cancer, hyperplasias,
restenosis, cardiac hypertrophy, immune disorders, fungal
disorders, and inflammation.
BACKGROUND OF THE INVENTION
[0003] Among the therapeutic agents used to treat cancer are the
taxanes and vinca alkaloids, which act on microtubules.
Microtubules are the primary structural element of the mitotic
spindle. The mitotic spindle is responsible for distribution of
replicate copies of the genome to each of the two daughter cells
that result from cell division. It is presumed that disruption of
the mitotic spindle by these drugs results in inhibition of cancer
cell division, and induction of cancer cell death. However,
microtubules form other types of cellular structures, including
tracks for intracellular transport in nerve processes. Because
these agents do not specifically target mitotic spindles, they have
side effects that limit their usefulness.
[0004] Improvements in the specificity of agents used to treat
cancer is of considerable interest because of the therapeutic
benefits which would be realized if the side effects associated
with the administration of these agents could be reduced.
Traditionally, dramatic improvements in the treatment of cancer are
associated with identification of therapeutic agents acting through
novel mechanisms. Examples of this include not only the taxanes,
but also the camptothecin class of topoisomerase 1 inhibitors. From
both of these perspectives, mitotic kinesins are attractive targets
for new anti-cancer agents.
[0005] Mitotic kinesins are enzymes essential for assembly and
function of the mitotic spindle, but are not generally part of
other microtubule structures, such as in nerve processes. Mitotic
kinesins play essential roles during all phases of mitosis. These
enzymes are "molecular motors" that transform energy released by
hydrolysis of ATP into mechanical force which drives the
directional movement of cellular cargoes along microtubules. The
catalytic domain sufficient for this task is a compact structure of
approximately 340 amino acids. During mitosis, kinesins organize
microtubules into the bipolar structure that is the mitotic
spindle. Kinesins mediate movement of chromosomes along spindle
microtubules, as well as structural changes in the mitotic spindle
associated with specific pleases of mitosis. Experimental
perturbation of mitotic kinesin function causes malformation or
dysfunction of the mitotic spindle, frequently resulting in cell
cycle arrest and cell death.
[0006] Among the mitotic kinesins which have been identified is
KSP. KSP belongs to an evolutionarily conserved kinesin subfamily
of plus end-directed microtubule motors that assemble into bipolar
homotetramers consisting of antiparallel homodimers. During mitosis
KSP associates with microtubules of the mitotic spindle.
Microinjection of antibodies directed against KSP into human cells
prevents spindle pole separation during prometaphase, giving rise
to monopolar spindles and causing mitotic arrest and induction of
programmed cell death. KSP and related kinesins in other,
non-human, organisms, bundle antiparallel microtubules and slide
them relative to one another, thus forcing the two spindle poles
apart. KSP may also mediate in anaphase B spindle elongation and
focussing of microtubules at the spindle pole.
[0007] Human KSP (also termed HsEg5) has been described (Blangy, et
al., Cell, 83:1159-69 (1995); Whitehead, et al., Arthritis Rheum.,
39:1635-42 (1996); Galgio et al., J. Cell Biol., 135:339-414
(1996); Blangy, et al., J Biol. Chem., 272:19418-24 (1997); Blangy,
et al., Cell Motil Cytoskeleton, 40:174-82 (1998); Whitehead and
Rattner, J. Cell Sci., 111:2551-61 (1998); Kaiser, et al., JBC
274:18925-31 (1999); GenBank accession numbers: X85137, NM004523
and U37426), and a fragment of the KSP gene (TRIP5) has been
described (Lee, et al., Mol Endocrinol., 9:243-54 (1995); GenBank
accession number L403 72). Xenopus KSP homologs (Eg5), as well as
Drosophila KLP61 F/KRP1 30 have been reported.
[0008] Mitotic kinesins, including KSP, are attractive targets for
the discovery and development of novel antimitotic
chemotherapeutics. Accordingly, it is an object of the present
invention to provide compounds, compositions and methods useful in
the inhibition of KSP.
SUMMARY OF THE INVENTION
[0009] In accordance with the objects outlined above, the present
invention provides compounds that can be used to treat cellular
proliferative diseases The compounds are KSP inhibitors,
particularly human KSP inhibitors. The present invention also
provides compositions comprising such compounds, and methods
utilizing such compounds or compositions, which can be used to
treat cellular proliferative diseases
[0010] In one aspect, the invention relates to methods for treating
cellular proliferative diseases, and for treating disorders by
inhibiting the activity of KSP. The methods employ compounds
represented by Formula I: ##STR1## wherein,
[0011] R.sub.1 is chosen from hydrogen, optionally substituted
alkyl, optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, and optionally substituted
heteroaralkyl;
[0012] R.sub.2 and R.sub.2' are independently chosen from hydrogen,
optionally substituted alkyl, optionally substituted alkoxy,
optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, and optionally substituted
heteroaralkyl; or R.sub.2 and R.sub.2' taken together form an
optionally substituted 3- to 7-membered ring;
[0013] R.sub.12 is selected from the group consisting of optionally
substituted imidazolyl, optionally substituted imidazolinyl,
--NHR.sub.4; --N(R.sub.4)(COR.sub.3);
--N(R.sub.4)(SO.sub.2R.sub.3a); and
--N(R.sub.4)(CH.sub.2R.sub.3b);
[0014] R.sub.3 is chosen from hydrogen, optionally substituted
alkyl, optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, optionally substituted
heteroaralkyl, R.sub.15O-- and R.sub.17--NH--;
[0015] R.sub.3a is chosen from optionally substituted alkyl,
optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, optionally substituted
heteroaralkyl, and R.sub.17--NH--;
[0016] R.sub.3b is chosen from hydrogen, optionally substituted
alkyl, optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, and optionally substituted
heteroaralkyl;
[0017] R.sub.4 is chosen from hydrogen, optionally substituted
alkyl, optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heterocyclyl, and optionally substituted
heteroaralkyl;
[0018] R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are independently
chosen from hydrogen, optionally substituted alkyl, optionally
substituted alkoxy, halogen, hydroxyl, nitro, cyano, dialkylamino,
alkylsulfonyl, alkylsulfonamido, alkylthio, carboxyalkyl,
carboxamido, aminocarbonyl, optionally substituted aryl and
optionally substituted heteroaryl;
[0019] R.sub.15 is chosen from optionally substituted alkyl,
optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, and optionally substituted
heteroaralkyl; and
[0020] R.sub.17 is chosen from hydrogen, optionally substituted
alkyl, optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, and optionally substituted
heteroaralkyl;
[0021] (Formula I including single stereoisomers and mixtures of
stereoisomers);
[0022] a pharmaceutically acceptable salt of a compound of Formula
I;
[0023] a pharmaceutically acceptable solvate of a compound of
Formula I; or
[0024] a pharmaceutically acceptable solvate of a pharmaceutically
acceptable salt of a compound of Formula I.
[0025] In one aspect, the invention relates to methods for treating
cellular proliferative diseases and other disorders that can be
treated by inhibiting KSP by the administration of a
therapeutically effective amount of a compound of Formula I; a
pharmaceutically acceptable salt of a compound of Formula I; a
pharmaceutically acceptable solvate of a compound of Formula I; or
a pharmaceutically acceptable solvate of a pharmaceutically
acceptable salt of a compound of Formula I. Such diseases and
disorders include cancer, hyperplasia, restenosis, cardiac
hypertrophy, immune disorders, fungal disorders and inflammation.
It will be understood that the method can employ one or more of the
foregoing compounds.
[0026] In another aspect, the invention relates to compounds useful
in inhibiting KSP kinesin. The compounds are selected from
compounds having tile structures shown above in Formula I;
pharmaceutically acceptable salts of a compound of Formula I;
pharmaceutically acceptable solvates of a compound of Formula I;
and pharmaceutically acceptable solvates of a pharmaceutically
acceptable salt of a compound of Formula I. The invention also
relates to pharmaceutical compositions comprising: a
therapeutically effective amount of a compound of Formula I; a
pharmaceutically acceptable salt of a compound of Formula I; a
pharmaceutically acceptable solvate of a compound of Formula I; or
a pharmaceutically acceptable solvate of a pharmaceutically
acceptable salt of a compound of Formula I; and one or more
pharmaceutical excipients. It will be understood that the
compositions can employ one or more of the foregoing compounds. In
another aspect, the composition further comprises a
chemotherapeutic agent other than a compound of the present
invention.
[0027] In an additional aspect, the present invention provides
methods of screening for compounds that will bind to a KSP kinesin,
for example compounds that will displace or compete with the
binding of a compound of the invention. The methods comprise
combining a labeled compound of the invention, a KSP kinesin, and
at least one candidate assent and determining the binding of the
candidate agent to the KSP kinesin.
[0028] In a further aspect, the invention provides methods of
screening for modulators of KSP kinesin activity. The methods
comprise combining a compound of the invention, a KSP kinesin, and
at least one candidate agent and determining the effect of the
candidate agent on the KSP kinesin activity.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
[0029] As used in the present specification, the following words
and phrases are generally intended to have the meanings as set
forth below, except to the extent that the context in which they
are used indicates otherwise. The following abbreviations and terms
have the indicated meanings throughout:
[0030] Ac=acetyl
[0031] BNB=4-bromomethyl-3-nitrobenzoic acid
[0032] Boc=t-butyloxy carbonyl
[0033] Bu=butyl
[0034] c-=cyclo
[0035] CBZ=carbobenzoxy=benzyloxycarbonyl
[0036] DBU=diazabicyclo[5.4.0]undec-7-ene
[0037] DCM=dichloromethane=methylene chloride=CH.sub.2Cl.sub.2
[0038] DCE=dichloroethane
[0039] DEAD=diethyl azodicarboxylate
[0040] DIC=diisopropylcarbodiimide
[0041] DIEA=N,N-diisopropylethylamine
[0042] DMAP=4-N,N-dimethylaminopyridine
[0043] DMF=N,N-dimethylformamide
[0044] DMSO=dimethyl sulfoxide
[0045] DVB=1,4-divinylbenzene
[0046] EEDQ=2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
[0047] Et=ethyl
[0048] Fmoc=9-fluorenylmethoxycarbonyl
[0049] GC=gas chromatography
[0050] HATU=O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
[0051] HMDS=hexamethyldisilazane
[0052] HOAc=acetic acid
[0053] HOBt=hydroxybenzotriazole
[0054] Me=methyl
[0055] mesyl=methanesulfonyl
[0056] MTBE=methyl t-butyl ether
[0057] NMO=N-methylmorpholine oxide
[0058] PEG=polyethylene glycol
[0059] Ph=phenyl
[0060] PhOH=phenol
[0061] PfP=pentafluorophenol
[0062] Pht=phthalyl
[0063] PPTS=pyridinium p-toluenesulfonate
[0064] Py=pyridine
[0065] PyBroP=bromo-tris-pyrrolidino-phosphonium
hexafluorophosphate
[0066] rt=room temperature
[0067] sat'd=saturated
[0068] s-=secondary
[0069] t-=tertiary
[0070] TBDMS=t-butyldimethylsilyl
[0071] TES=triethylsilyl
[0072] TFA=trifluoroacetic acid
[0073] THF=tetrahydrofuran
[0074] TMOF=trimethyl orthoformate
[0075] TMS=trimethylsilyl
[0076] tosyl=p-toluenesulfonyl
[0077] Trt=triphenylmethyl
[0078] Alkyl is intended to include linear, branched, or cyclic
aliphatic hydrocarbon structures and combinations thereof, which
structures may be saturated or unsaturated. Lower-alkyl refers to
alkyl groups of from 1 to 5 carbon atoms, preferably from 1 to 4
carbon atoms. Examples of lower-alkyl groups include methyl-,
ethyl-, propyl-, isopropyl-, butyl-, s- and t-butyl and tile like.
Preferred alkyl groups are those of C.sub.20 or below. More
preferred alkyl groups are those of C.sub.13 or below. Cycloalkyl
is a subset of alkyl and includes cyclic aliphatic hydrocarbon
groups of from 3 to 13 carbon atoms. Examples of cycloalkyl groups
include c-propyl-, c-butyl, c-pentyl-, norbornyl-, adamantyl and
the like. Cycloalkyl-alkyl- is another subset of alkyl and refers
to cycloalkyl attached to the parent structure through a non-cyclic
alkyl-. Examples of cycloalkyl-alkyl- include cyclohexylmethyl-,
cyclopropylmethyl-, cyclohexylpropyl-, and the like. In this
application, alkyl includes alkanyl-, alkenyl and alkynyl residues;
it is intended to include vinyl-, allyl-, isoprenyl and the like.
When an alkyl residue having a specific number of carbons is named,
all geometric isomers having that number of carbons are intended to
be encompassed; thus, for example, "butyl" is meant to include
n-butyl-, sec-butyl-, isobutyl and t-butyl-; "propyl" includes
n-propyl-, isopropyl-, and c-propyl-.
[0079] Alkylene-, alkenylene-, and alkynylene- are other subsets of
alkyl-, including the same residues as alkyl-, but having two
points of attachment within a chemical structure. Examples of
alkylene include ethylene (--CH.sub.2CH.sub.2--), propylene
(--CH.sub.2CH.sub.2CH.sub.2--), dimethylpropylene
(--CH.sub.2C(CH.sub.3).sub.2CH.sub.2--) and cyclohexylpropylene
(--CH.sub.2CH.sub.2CH(C.sub.6H.sub.13)--). Likewise, examples of
alkenylene include ethenylene (--CH.dbd.CH--), propenylene
(--CH.dbd.CH--CH.sub.2--), and cyclohexylpropenylene
(--CH.dbd.CHCH(C.sub.6H.sub.13)--). Examples of alkynylene include
ethynylene (--C.dbd.C--) and propynylene
(--CH.dbd.CH--CH.sub.2--).
[0080] Cycloalkenyl is a subset of alkyl and includes unsaturated
cyclic hydrocarbon groups of from 3 to 13 carbon atoms. Examples of
cycloalkenyl groups include c-hexenyl-, c-pentenyl and the
like.
[0081] Alkoxy or alkoxyl refers to an alkyl group, preferably
including from 1 to 8 carbon atoms, of a straight, branched, or
cyclic configuration, or a combination thereof, attached to the
parent structure through an oxygen (i.e., the group alkyl-O--).
Examples include methoxy-, ethoxy-, propoxy-, isopropoxy-,
cyclopropyloxy-, cyclohexyloxy- and the like. Lower-alkoxy refers
to alkoxy groups containing one to four carbons.
[0082] Acyl refers to groups of from 1 to 8 carbon atoms of a
straight, branched, or cyclic configuration or a combination
thereof attached to the parent structure through a carbonyl
functionality. Such groups may be saturated or unsaturated, and
aliphatic or aromatic. One or more carbons in the acyl residue may
be replaced by oxygen, nitrogen (e.g., carboxamido), or sulfur as
long as the point of attachment to the parent remains at the
carbonyl. Examples include acetyl-, benzoyl-, propionyl-,
isobutyryl-, oxalyl-, t-butoxycarbonyl-, benzyloxycarbonyl,
morpholinylcarbonyl, and the like. Lower-acyl refers to acyl groups
containing one to four carbons.
[0083] Amino refers to the group --NH.sub.2. The term "substituted
amino" refers to the group --NHR or --NRR where each R is
independently selected from the group: optionally substituted
alkyl-, optionally substituted alkoxy, optionally substituted
aminocarbonyl-, optionally substituted aryl-, optionally
substituted heteroaryl-, optionally substituted heterocyclyl-,
acyl-, alkoxycarbonyl-, sulfanyl-, sulfinyl and sulfonyl-, e.g.,
diethylamino, methylsulfonylamino, furanyl-oxy-sulfonamino.
[0084] Aminocarbonyl- refers to the group --NR.sup.cCOR.sup.b,
--NR.sup.cCO.sub.2R.sup.a, or --NR.sup.cCONR.sup.bR.sup.c,
where
[0085] R.sup.1 is optionally substituted C.sub.1-C.sub.6 alkyl-,
aryl-, heteroaryl-, aryl-C.sub.1-C.sub.4 alkyl-, or
heteroaryl-C.sub.1-C.sub.4 alkyl- group;
[0086] R.sup.b is H or optionally substituted C.sub.1-C.sub.6
alkyl-, aryl-, heteroaryl-, aryl-C.sub.1-C.sub.4 alkyl-, or
heteroaryl-C.sub.1-C.sub.4 alkyl- group; and
[0087] R.sup.c is hydrogen, alkyl-; aryl- or heteroaryl-; and where
each optionally substituted R.sup.b group is independently
unsubstituted or substituted with one or more substituents
independently selected from C.sub.1-C.sub.4 alkyl-, aryl-,
heterocyclyl-, aryl-C.sub.1-C.sub.4 alkyl-,
heteroaryl-C.sub.1-C.sub.4 alkyl-, C.sub.1-C.sub.4 haloalkyl-,
--OC.sub.1-C.sub.4 alkyl, --OC.sub.1-C.sub.4 alkylphenyl,
--C.sub.1-C.sub.4 alkyl-OH, --OC.sub.1-C.sub.4 haloalkyl, halogen,
--OH, --NH.sub.2, --NR.sup.cC(NR.sup.b)(NR.sup.bR.sup.c) (i.e.
guanidine), --NR.sup.cCR.sup.bNR.sup.bR.sup.c,
--CNR.sup.cNR.sup.bR.sup.c, --C.sub.1-C.sub.4 alkyl-NH.sub.2,
--N(C.sub.1-C.sub.4 alkyl)(C.sub.1-C.sub.4 alkyl),
--NH(C.sub.1-C.sub.4 alkyl), --N(C.sub.1-C.sub.4
alkyl)(C.sub.1-C.sub.4 alkylphenyl), --NH(C.sub.1-C.sub.4
alkylphenyl), cyano, nitro, oxo (as a substituent for heteroaryl),
--CO.sub.2H, --C(O)OC.sub.1-C.sub.4 alkyl-, --CON(C.sub.1-C.sub.4
alkyl)(C.sub.1-C.sub.4 alkyl), --CONH(C.sub.1-C.sub.4 alkyl),
--CONH.sub.2, --NHC(O)(C.sub.1-C.sub.4 alkyl), --NHC(O)(phenyl),
--N(C.sub.1-C.sub.4 alkyl)C(O)(C.sub.1-C.sub.4 alkyl),
--N(C.sub.1-C.sub.4 alkyl)C(O)(phenyl), --C(O)C.sub.1-C.sub.4
alkyl-, --C(O)C.sub.1-C.sub.4 phenyl-, --C(O)C.sub.1-C.sub.4
haloalkyl-, --OC(O)C.sub.1-C.sub.4 alkyl-,
--SO.sub.2(C.sub.1-C.sub.4 alkyl), --SO.sub.2(phenyl),
--SO.sub.2(C.sub.1-C.sub.4 haloalkyl), --SO.sub.2NH.sub.2,
--SO.sub.2NH(C.sub.1-C.sub.4 alkyl), --SO.sub.2NH(phenyl),
--NHSO.sub.2(C.sub.1-C.sub.4 alkyl), --NHSO.sub.2(phenyl), and
--NHSO.sub.2(C.sub.1-C.sub.4 haloalkyl).
[0088] Antimitotic refers to a drug for inhibiting or preventing
mitosis, for example, by causing metaphase arrest. Some antitumour
drugs block proliferation and are considered antimitotics.
[0089] Aryl and heteroaryl mean a 5- or 6-membered aromatic or
heteroaromatic ring containing 0 or 1-4 heteroatoms, respectively,
selected from O, N, or S; a bicyclic 9- or 10-membered aromatic or
heteroaromatic ring system containing 0 or 1-4 (or more)
heteroatoms, respectively, selected from O, N, or S; or a tricyclic
12- to 14-membered aromatic or heteroaromatic ring system
containing 0 or 1-4 (or more) heteroatoms, respectively, selected
from O, N, or S. The aromatic 6- to 14-membered carbocyclic rings
include, e.g., phenyl-, naphthyl-, indanyl-, tetralinyl-, and
fluorenyl and the 5- to 10-membered aromatic heterocyclic rings
include, e.g., imidazolyl-, pyridinyl-, indolyl-, thienyl-,
benzopyranonyl-, thiazolyl-, furanyl-, benzimidazolyl-,
quinolinyl-, isoquinolinyl-, quinoxalinyl-, pyrimidinyl-,
pyrazinyl-, tetrazolyl and pyrazolyl-.
[0090] Aralkyl- refers to a residue in which an aryl moiety is
attached to the parent structure via an alkyl residue. Examples
include benzyl-, phenethyl-, phenylvinyl-, phenylallyl and the
like. Heteroaralkyl- refers to a residue in which a heteroaryl
moiety is attached to the parent structure via an alkyl residue.
Examples include furanylmethyl-, pyridinylmethyl-, pyrimidinylethyl
and the like.
[0091] Aralkoxy- refers to the group --O-aralkyl. Similarly,
heteroaralkoxy- refers to the group --O-heteroaralkyl-; aryloxy-
refers to the group --O-aryl-; acyloxy- refers to the group
--O-acyl-; heteroaryloxy- refers to the group --O-heteroaryl-; and
heterocyclyloxy- refers to the group --O-heterocyclyl (i.e.,
aralkyl-, heteroaralkyl-, aryl-, acyl-, heterocyclyl-, or
heteroaryl is attached to the parent structure through an
oxygen).
[0092] Carboxyalkyl- refers to the group -alkyl-COOH.
[0093] Carboxamido refers to the group --CONR.sup.bR.sup.c,
where
[0094] R.sup.b is H or optionally substituted C.sub.1-C.sub.6
alkyl-, aryl-, heteroaryl-, aryl-C.sub.1-C.sub.4 alkyl-, or
heteroaryl-C.sub.1-C.sub.4 alkyl- group; and
[0095] R.sup.c is hydrogen, alkyl-; aryl- or heteroaryl-; and
[0096] where each optionally substituted R.sup.b group is
independently unsubstituted or substituted with one or more
substituents independently selected from C.sub.1-C.sub.4 alkyl-,
aryl-, heterocyclyl-, aryl-C.sub.1-C.sub.4 alkyl-,
heteroaryl-C.sub.1-C.sub.4 alkyl-, C.sub.1-C.sub.4 haloalkyl-,
--OC.sub.1-C.sub.4 alkyl-, --OC.sub.1-C.sub.4 alkylphenyl-,
--C.sub.1-C.sub.4 alkyl-OH, --OC.sub.1-C.sub.4 haloalkyl-, halogen,
--OH, --NH.sub.2, --C.sub.1-C.sub.4 alkyl-NH.sub.2,
--N(C.sub.1-C.sub.4 alkyl)(C.sub.1-C.sub.4 alkyl),
--NH(C.sub.1-C.sub.4 alkyl), --N(C.sub.1-C.sub.4
alkyl)(C.sub.1-C.sub.4 alkylphenyl), --NH(C.sub.1-C.sub.4
alkylphenyl), cyano, nitro, oxo (as a substituent for heteroaryl),
--CO.sub.2H, --C(O)OC.sub.1-C.sub.4 alkyl-, --CON(C.sub.1-C.sub.4
alkyl)(C.sub.1-C.sub.4 alkyl), --CONH(C.sub.1-C.sub.4 alkyl),
--CONH.sub.2, --NHC(O)(C.sub.1-C.sub.4 alkyl), --NHC(O)(phenyl),
--N(C.sub.1-C.sub.4 alkyl)C(O)(C.sub.1-C.sub.4 alkyl),
--N(C.sub.1-C.sub.4 alkyl)C(O)(phenyl), --C(O)C.sub.1-C.sub.4
alkyl-, --C(O)C.sub.1-C.sub.4 phenyl-, --C(O)C.sub.1-C.sub.4
haloalkyl-, --OC(O)C.sub.1-C.sub.4 alkyl-,
--SO.sub.2(C.sub.1-C.sub.4 alkyl), --SO.sub.2(phenyl),
--SO.sub.2(C.sub.1-C.sub.4 haloalkyl), --SO.sub.2NH.sub.2,
--SO.sub.2NH(C.sub.1-C.sub.4 alkyl), --SO.sub.2NH(phenyl),
--NHSO.sub.2(C.sub.1-C.sub.4 alkyl), --NHSO.sub.2(phenyl), and
--NHSO.sub.2(C.sub.1-C.sub.4 haloalkyl). Carboxamido is meant to
include carbamoyl-; lower-alkyl carbamoyl-; benzylcarbamoyl-;
phenylcarbamoyl-; methoxymethyl-carbamoyl-; and the like.
[0097] Halogen or halo refers to fluorine, chlorine, bromine or
iodine. Fluorine, chlorine and bromine are preferred. Dihaloaryl-,
dihaloalkyl-, trihaloaryl etc. refer to aryl and alkyl substituted
with the designated plurality of halogens (here, 2, 2 and 3,
respectively), but not necessarily a plurality of the same halogen;
thus 4-chloro-3-fluorophenyl is within the scope of
dihaloaryl-.
[0098] Heterocyclyl means a cycloalkyl or aryl residue in which one
to foul of the carbons is replaced by a heteroatom such as oxygen,
nitrogen or sulfur. Examples of heterocycles that fall within the
scope of the invention include azetidinyl-, imidazolinyl-,
pyrrolidinyl-, pyrazolyl-, pyrrolyl-, indolyl-, quinolinyl-,
isoquinolinyl-, tetrahydroisoquinolinyl-, benzofuranyl-,
benzodioxanyl-, benzodioxyl (commonly referred to as
methylenedioxyphenyl-, when occurring as a substituent),
tetrazolyl-, morpholinyl-, thiazolyl-, pyridinyl-, pyridazinyl-,
piperidinyl-, pyrimidinyl-, thienyl-, furanyl-, oxazolyl-,
oxazolinyl-, isoxazolyl-, dioxanyl-, tetrahydrofuranyl and the
like. "N-heterocyclyl" refers to a nitrogen-containing heterocycle.
The term heterocyclyl encompasses heteroaryl-, which is a subset of
heterocyclyl-. Examples of N-heterocyclyl residues include
azetidinyl-, 4-morpholinyl-, 4-thiomorpholinyl-, 1-piperidinyl-,
1-pyrrolidinyl-, 3-thiazolidinyl-, piperazinyl and
4-(3,4-dihydrobenzoxazinyl). Examples of substituted heterocyclyl
include 4-methyl-1-piperazinyl and 4-benzyl-1-piperidinyl-.
[0099] A leaving group or atom is any group or atom that will,
under the reaction conditions, cleave from the stating material,
thus promoting reaction at a specified site. Suitable examples of
such groups unless otherwise specified are halogen atoms, mesyloxy,
p-nitrobenzensulphonyloxy and tosyloxy groups.
[0100] Optional or optionally means that the subsequently described
event or circumstance may or may not occur, and that the
description includes instances where said event or circumstances
occurs and instances in which it does not. For example, "optionally
substituted alkyl" includes "alkyl" and "substituted alkyl" as
defined herein. It wilt be understood by those skilled in the art
with respect to any group containing one or more substituents that
such groups are not intended to introduce any substitution or
substitution patterns that are sterically impractical and/or
synthetically non-feasible and/or inherently unstable.
[0101] Substituted alkoxy refers to alkoxy wherein the alkyl
constituent is substituted (i.e., --O-(substituted alkyl)). One
suitable substituted alkoxy group is "polyalkoxy" or
--O-(optionally substituted alkylene)-(optionally substituted
alkoxy), and includes groups such as --OCH.sub.2CH.sub.2OCH.sub.3,
and residues of glycol ethers such as polyethyleneglycol, and
--O(CH.sub.2CH.sub.2O).sub.xCH.sub.3, where x is an integer of
about 2-20, preferably about 2-10, and more preferably about 2-5.
Another suitable substituted alkoxy group is hydroxyalkoxy or
--OCH.sub.2(CH.sub.2).sub.yOH, where y is an integer of about 1-10,
preferably about 1-4.
[0102] Substituted- alkyl-, aryl-, and heteroaryl- refer
respectively to alkyl-, aryl-, and heteroaryl wherein one or more
(up to about 5, preferably up to about 3) hydrogen atoms are
replaced by a substituent independently selected from the group:
--R.sup.a, --OR.sup.b, --O(C.sub.1-C.sub.2 alkyl)O-- (as an aryl
substituent), --SR.sup.b, --NR.sup.bR.sup.c,
--NR.sup.cC(NR.sup.b)(NR.sup.bR.sup.c) (i.e, guanidine),
--NR.sup.cCR.sup.bNR.sup.bR.sup.c, --CNR.sup.cNR.sup.bR.sup.c,
halogen, cyano, nitro, --COR.sup.b, --CO.sub.2R.sup.b,
--CONR.sup.bR.sup.c, --OCOR.sup.b, --OCO.sub.2R.sup.a,
--OCONR.sup.bR.sup.c, --NR.sup.cCOR.sup.b, --NR.sup.cCOR.sup.b,
--NR.sup.cCO.sub.2R.sup.a, --NR.sup.cCONR.sup.bR.sup.c,
--CO.sub.2R.sup.b, --CONR.sup.bR.sup.c, --NR.sup.cCOR.sup.b,
--SOR.sup.a, --SO.sub.2R.sup.a, --SO.sub.2NR.sup.bR.sup.c, and
--NR.sup.cSO.sub.2R.sup.a,
[0103] where R.sup.a is an optionally substituted C.sub.1-C.sub.6
alkyl-, aryl-, heteroaryl-, aryl-C.sub.1-C.sub.4 alkyl-, or
heteroaryl-C.sub.1-C.sub.4 alkyl- group,
[0104] R.sup.b is H or optionally substituted C.sub.1-C.sub.6
alkyl-, aryl-, heteroaryl-, aryl-C.sub.1-C.sub.4 alkyl-, or
heteroaryl-C.sub.1-C.sub.4 alkyl- group;
[0105] R.sup.c is hydrogen, alkyl-; aryl- or heteroaryl-; and where
each optionally substituted R.sup.a group and R.sup.b group is
independently unsubstituted or substituted with one or more
substituents independently selected from C.sub.1-C.sub.4 alkyl-,
aryl-, heterocyclyl-, aryl-C.sub.1-C.sub.4 alkyl-,
heteroaryl-C.sub.1-C.sub.4 alkyl-, C.sub.1-C.sub.4 haloalkyl-,
--OC.sub.1-C.sub.4 alkyl-, --OC.sub.1-C.sub.4 alkylphenyl-,
--C.sub.1-C.sub.4 alkyl-OH, --OC.sub.1-C.sub.4 haloalkyl-, halogen,
--OH, --NH.sub.2, --C.sub.1-C.sub.4 alkyl-NH.sub.2,
--N(C.sub.1-C.sub.4 alkyl)(C.sub.1-C.sub.4 alkyl),
--NH(C.sub.1-C.sub.4 alkyl), --N(C.sub.1-C.sub.4
alkyl)(C.sub.1-C.sub.4 alkylphenyl), --NH(C.sub.1-C.sub.4
alkylphenyl), cyano, nitro, oxo (as a substituent for heteroaryl),
--CO.sub.2H, --C(O)OC.sub.1-C.sub.4 alkyl-, --CON(C.sub.1-C.sub.4
alkyl)(C.sub.1-C.sub.4 alkyl), --CONH(C.sub.1-C.sub.4 alkyl),
--CONH.sub.2, --NHC(O)(C.sub.1-C.sub.4 alkyl), --NHC(O)(phenyl),
--N(C.sub.1-C.sub.4 alkyl)C(O)(C.sub.1-C.sub.4 alkyl),
--N(C.sub.1-C.sub.4 alkyl)C(O)(phenyl), --C(O)C.sub.1-C.sub.4
alkyl-, --C(O)C.sub.1-C.sub.4 phenyl-, --C(O)C.sub.1-C.sub.4
haloalkyl-, --OC(O)C.sub.1-C.sub.4 alkyl-,
--SO.sub.2(C.sub.1-C.sub.4 alkyl), --SO.sub.2(phenyl),
--SO.sub.2(C.sub.1-C.sub.4 haloalkyl), --SO.sub.2NH.sub.2,
--SO.sub.2NH(C.sub.1-C.sub.4 alkyl), --SO.sub.2NH(phenyl),
--NHSO.sub.2(C.sub.1-C.sub.4 alkyl), --NHSO.sub.2(phenyl), and
--NHSO.sub.2(C.sub.1-C.sub.4 haloalkyl).
[0106] Sulfanyl refers to the groups: --S-(optionally substituted
alkyl), --S-(optionally substituted aryl), --S-(optionally
substituted heteroaryl), and --S-(optionally substituted
heterocyclyl).
[0107] Sulfinyl refers to the groups: --S(O)--H, --S(O)-(optionally
substituted alkyl), --S(O)-optionally substituted aryl),
--S(O)-(optionally substituted heteroaryl), --S(O)-(optionally
substituted heterocyclyl); and --S(O)-(optionally substituted
amino).
[0108] Sulfonyl refers to the groups: --S(O.sub.2)--H,
--S(O.sub.2)-(optionally substituted alkyl),
--S(O.sub.2)-optionally substituted aryl), --S(O.sub.2)-(optionally
substituted heteroaryl), --S(O.sub.2)-(optionally substituted
heterocyclyl), --S(O.sub.2)-(optionally substituted alkoxy),
--S(O.sub.2)-optionally substituted aryloxy),
--S(O.sub.2)-(optionally substituted heteroaryloxy),
--S(O.sub.2)-(optionally substituted heterocyclyloxy); and
--S(O.sub.2)-(optionally substituted amino).
[0109] Pharmaceutically acceptable salts refers to those salts that
retain the biological effectiveness of the free compound and that
are not biologically undesirable or unsuitable for pharmaceutical
use, formed with a suitable acid or base, and includes
pharmaceutically acceptable acid addition salts and base addition
salts. Pharmaceutically acceptable acid addition salts include
those derived from inorganic acids such as hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and
the like, and those derived from organic acids such as acetic acid,
propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic
acid, malonic acid, succinic acid, fumaric acid, tartaric acid,
citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid and the like.
[0110] Pharmaceutically acceptable base addition salts include
those derived from inorganic bases such as sodium, potassium,
lithium, ammonium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum salts and the like. Particular embodiments are
the ammonium, potassium, sodium, calcium, and magnesium salts. Base
addition salts also include those derived from pharmaceutically
acceptable organic non-toxic bases, including salts of primary,
secondary, and tertiary amines, substituted amines including
naturally occurring substituted amines, cyclic amines and basic ion
exchange resins, such as isopropylamine, trimethylamine,
diethylamine, triethylamine, tripropylamine, and ethanolamine.
[0111] Protecting group has the meaning conventionally associated
with it in organic synthesis, i.e. a group that selectively blocks
one or more reactive sites in a multifunctional compound such that
a chemical reaction can be carried out selectively on another
unprotected reactive site and such that the group can readily be
removed after the selective reaction is complete. A variety of
protecting groups are disclosed, for example, in T. H. Greene and
P. G. M. Wuts, Protective Groups in Organic Synthesis, Third
Edition, John Wiley & Sons, New York (1999), which is
incorporated herein by reference in its entirety. For example, a
hydroxy protected form is where at least one of the hydroxyl groups
present in a compound is protected with a hydroxy protecting group.
Likewise, amities and other reactive groups may similarly be
protected.
[0112] Solvate refers to the compound formed by the interaction of
a solvent and a compound of Formula I or salt thereof, and as will
be understood by those skilled in the art, is the compound or salt
with which a solvent is incorporated, for example, into the crystal
structure. Suitable solvates of the compounds of the Formula I or a
salt thereof are those formed with a pharmaceutically acceptable
solvent, including hydrates (i.e., wherein the solvent is
water).
[0113] Many of the compounds described herein contain one or more
asymmetric centers (e.g. the carbon to which R.sub.2 and R.sub.2'
are attached where R.sub.2 differs from R.sub.2') and may thus give
rise to enantiomers, diastereomers, and other stereoisomeric forms
that may be defined, in terms of absolute stereochemistry, as (R)--
or (S)--. The present invention is meant to include all such
possible isomers, including racemic mixtures, optically pure forms
and intermediate mixtures. Optically active (R)-- and (S)-isomers
may be prepared using chiral synthons or chiral reagents, or
resolved using conventional techniques. When the compounds
described herein contain olefinic double bonds or other centers of
geometric asymmetry, and unless specified otherwise, it is intended
that the compounds include both E and Z geometric isomers.
Likewise, all tautomeric forms and rotational isomers are also
intended to be included.
[0114] When desired, the R-- and S-isomers may be resolved by
methods known to those skilled in the art, for example by formation
of diastereoisomeric salts or complexes which may be separated, for
example, by crystallization; via formation of diastereoisomeric
derivatives which may be separated, for example, by
crystallization, gas-liquid or liquid chromatography; selective
reaction of one enantiomer with an enantiomer-specific reagent, for
example enzymatic oxidation or reduction, followed by separation of
the modified and unmodified enantiomers; or gas-liquid or liquid
chromatography in a chiral environment, for example on a chiral
support, such as silica with a bound chiral ligand or in the
presence of a chiral solvent. It will be appreciated that where the
desired enantiomer is converted into another chemical entity by one
of the separation procedures described above, a further step may be
required to liberate the desired enantiomeric form. Alternatively,
specific enantiomer may be synthesized by asymmetric synthesis
using optically active reagents, substrates, catalysts or solvents,
or by converting one enantiomer to the other by asymmetric
transformation.
Compounds of the Present Invention
[0115] The present invention is directed to a class of novel
compounds, that can be described as isoquinolone derivatives, that
are inhibitors of one or more mitotic kinesins. By inhibiting
mitotic kinesins, but not other kinesins (e.g., transport
kinesins), specific inhibition of cellular proliferation is
accomplished. While not intending to be bound by any theory, the
present invention capitalizes on the finding that perturbation of
mitotic kinesin function causes malformation or dysfunction of
mitotic spindles, frequently resulting in cell cycle arrest and
cell death. According to one embodiment of the invention, the
compounds described herein inhibit the mitotic kinesin, KSP,
particularly human KSP. In another embodiment, the compounds
inhibit the mitotic kinesin, KSP, as well as modulating one or more
of the human mitotic kinesins selected from the group consisting of
HSET (see, U.S. Pat. No. 6,361,993, which is incorporated herein by
reference); MCAK (see, U.S. Pat. No. 6,331,424, which is
incorporated herein by reference); CENP-E (see, PCT Publication No.
WO 99/13061, which is incorporated herein by reference); Kif4 (see,
U.S. Pat. No. 6,440,684, which is incorporated herein by
reference); MKLP1 (see, U.S. Pat. No. 6,448,025, which is
incorporated herein by reference); Kif15 (see, U.S. Pat. No.
6,355,466, which is incorporated herein by reference); Kid (see,
U.S. Pat. No. 6,387,644, which is incorporated herein by
reference); Mpp1, CMKrp, Kin1-3 (see, U.S. Pat. No. 6,461,855,
which is incorporated herein by reference); Kip3a (see, PCT
Publication No. WO 01/96593, which is incorporated herein by
reference); Kip3d (see, U.S. Pat. No. 6,492,151, which is
incorporated herein by reference); and RabK6.
[0116] The methods of inhibiting a mitotic kinesin comprise
contacting an inhibitor of the invention with a kinesin,
particularly a human kinesin, more particularly, human KSP or
fragments and variants thereof. The inhibition can be of the ATP
hydrolysis activity of the KSP kinesin and/or the mitotic spindle
formation activity, such that the mitotic spindles are disrupted.
Meiotic spindles may also be disrupted.
[0117] The present invention provides inhibitors of mitotic
kinesins, in particular KSP and especially human KSP, for the
treatment of disorders associated with cell proliferation. The
compounds, compositions and methods described herein can differ in
their selectivity and are used to treat diseases of cellular
proliferation, including, but not limited to cancer, hyperplasias,
restenosis, cardiac hypertrophy, immune disorders, fungal disorders
and inflammation.
[0118] Accordingly, the present invention relates to methods
employing compounds represented by Formula I: ##STR2## wherein,
[0119] R.sub.1 is chosen from hydrogen, optionally substituted
alkyl, optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, and optionally substituted
heteroaralkyl;
[0120] R.sub.2 and R.sub.2' are independently chosen from hydrogen,
optionally substituted alkyl, optionally substituted alkoxy,
optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, and optionally substituted
heteroaralkyl; or R.sub.2 and R.sub.2' taken together form an
optionally substituted 3- to 7-membered ring;
[0121] R.sub.12 is selected from the group consisting of optionally
substituted imidazolyl, optionally substituted imidazolinyl,
--NHR.sub.4; --N(R.sub.4)(COR.sub.3);
--N(R.sub.4)(SO.sub.2R.sub.3a); and
--N(R.sub.4)(CH.sub.2R.sub.3b);
[0122] R.sub.3 is chosen from hydrogen, optionally substituted
alkyl, optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, optionally substituted
heteroaralkyl, R.sub.15O-- and R.sub.17--NH--;
[0123] R.sub.3a is chosen from optionally substituted alkyl,
optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, optionally substituted
heteroaralkyl, and R.sub.17--NH--;
[0124] R.sub.3b is chosen from hydrogen, optionally substituted
alkyl, optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, and optionally substituted
heteroaralkyl;
[0125] R.sub.4 is chosen from hydrogen, optionally substituted
alkyl, optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heterocyclyl, and optionally substituted
heteroaralkyl;
[0126] R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are independently
chosen from hydrogen, optionally substituted alkyl, optionally
substituted alkoxy, halogen, hydroxyl, nitro, cyano, dialkylamino,
alkylsulfonyl, alkylsulfonamido, alkylthio, carboxyalkyl,
carboxamido, aminocarbonyl, optionally substituted aryl and
optionally substituted heteroaryl;
[0127] R.sub.15 is chosen from optionally substituted alkyl,
optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, and optionally substituted
heteroaralkyl; and
[0128] R.sub.17 is chosen from hydrogen, optionally substituted
alkyl, optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, and optionally substituted
heteroaralkyl;
[0129] including single stereoisomers and mixtures of
stereoisomers;
[0130] a pharmaceutically acceptable salt of a compound of Formula
I;
[0131] a pharmaceutically acceptable solvate of a compound of
Formula I;
[0132] or a pharmaceutically acceptable solvate of a
pharmaceutically acceptable salt of a compound of Formula I. In a
particular embodiment, the stereogenic center to which R.sub.2 and
R.sub.2' are attached is of the R configuration.
Nomenclature
[0133] The compounds of Formula I can be named and numbered in the
manner described below (e.g., using AutoNom version 2.1 in
ISIS-DRAW or ChemDraw). For example, the compound: ##STR3## i.e.,
the compound according to Formula I where R.sub.1 is benzyl-,
R.sub.2 is propyl (or i-propyl), R.sub.2' is hydrogen; R.sub.12 is
substituted imidazolinyl wherein R.sub.10 and R.sub.10' are methyl,
R.sub.11 and R.sub.11' are hydrogen and R.sub.14 is p-tolyl;
R.sub.5, R.sub.6, and R.sub.8 are hydrogen; and R.sub.7 is chloro
can be named
2-Benzyl-6-chloro-3-[1-(4,4-dimethyl-2-p-tolyl-4,5-dihydro-imidazol-1-yl)-
-2-methyl-propyl]-2H-isoquinolin-1-one.
[0134] Likewise, the compound: ##STR4## i.e., the compound
according to Formula I where R.sub.1 is benzyl-, R.sub.2 is propyl
(or i-propyl), R.sub.2' is hydrogen; R.sub.12 is
--N(R.sub.4)(COR.sub.3) wherein R.sub.4 is 3-aminopropyl- and
R.sub.3 is p-tolyl; R.sub.5, R.sub.6, and R.sub.8 are hydrogen; and
R.sub.7 is chloro can be named
N-(3-Amino-propyl)-N-[1-(2-benzyl-6-chloro-1-oxo-1,2-dihydro-isoquinolin--
3-yl)-2-methyl-propyl]-4-methyl-benzamide. Synthetic Reaction
Parameters
[0135] The compounds of Formula I can be prepared by following the
procedures described with reference to the Reaction Schemes
below.
[0136] Unless specified otherwise, the terms "solvent", "inert
organic solvent" or "inert solvent" mean a solvent inert under the
conditions of the reaction being described in conjunction therewith
[including, for example, benzene, toluene, acetonitrile,
tetrahydrofuran ("THF"), dimethylformamide ("DMF"), chloroform,
methylene chloride (or dichloromethane), diethyl ether, methanol,
pyridine and the like]. Unless specified to the contrary, the
solvents used in the reactions of the present invention are inert
organic solvents.
[0137] The term "q.s." means adding a quantity sufficient to
achieve a stated function, e.g., to bring a solution to the desired
volume (i.e., 100%).
[0138] In general, esters of carboxylic acids may be prepared by
conventional esterification procedures, for example alkyl esters
may be prepared by treating the required carboxylic acid with the
appropriate alkanol, generally under acidic conditions. Likewise,
amides may be prepared using conventional amidation procedures, for
example amides may be prepared by treating an activated carboxylic
acid with the appropriate amine. Alternatively, a lower-alkyl ester
such as a methyl ester of the acid may be treated with an amine to
provide the required amide, optionally in presence of
trimethylalluminium following the procedure described in
Tetrahedron Lett. 48, 4171-4173, (1977). Carboxyl groups may be
protected as alkyl esters, for example methyl esters, which esters
may be prepared and removed using conventional procedures, one
convenient method for converting carbomethoxy to carboxyl is to use
aqueous lithium hydroxide.
[0139] The salts and solvates of the compounds mentioned herein may
as required be produced by methods conventional in the art. For
example, if an inventive compound is an acid, a desired base
addition salt can be prepared by treatment of the free acid with an
inorganic or organic base, such as an amine (primary, secondary, or
tertiary); an alkali metal or alkaline earth metal hydroxide; or
the like. Illustrative examples of suitable salts include organic
salts derived from amino acids such as glycine and arginine;
ammonia; primary, secondary, and tertiary amines; such as
ethylenediamine, and cyclic amines, such as cyclohexylamine,
piperidine, morpholine, and piperazine; as well as inorganic salts
derived from sodium, calcium, potassium, magnesium, manganese,
iron, copper, zinc, aluminum, and lithium.
[0140] If a compound is a base, a desired acid addition salt may be
prepared by any suitable method known in the art, including
treatment of the free base with an inorganic acid, such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid, and the like, or with an organic acid, such as
acetic acid, maleic acid, succinic acid, mandelic acid, fumaric
acid, malonic acid, pyruvic acid, oxalic acid, (glycolic acid,
salicylic acid, pyranosidyl acid, such as (glucuronic acid or
galacturonic acid, alpha-hydroxy acid, such as citric acid or
tartaric acid, amino acid, such as aspartic acid or glutamic acid,
aromatic acid, such as benzoic acid or cinnanic acid, sulfonic
acid, such as p-toluenesulfonic acid, methanesulfonic acid,
ethanesulfonic acid, or the like.
[0141] Isolation and purification of the compounds and
intermediates described herein can be effected, if desired, by any
suitable separation or purification procedure such as, for example,
filtration, extraction, crystallization, column chromatography,
thin-layer chromatography or thick-layer chromatography, or a
combination of these procedures. Specific illustrations of suitable
separation and isolation procedures can be had by reference to the
examples hereinbelow. However, other equivalent separation or
isolation procedures can, of course, also be used.
Synthesis of the Compounds of Formula I
[0142] The compounds of Formula I can be prepared by following the
procedures described in U.S. patent application Ser. No. 10/366,828
and corresponding PCT Application No. US03/04713; and PCT
Publication Nos. WO 01/30768 and WO 01/98278, each of which is
incorporated herein by reference for all purposes, and with
reference to the Reaction Schemes below.
Brief Description of Reaction Schemes
[0143] Reaction Scheme 1 illustrates a synthesis of an intermediate
(i.e., compounds of Formula 113) in the synthesis of compounds of
Formula I.
[0144] Reaction Scheme 2 illustrates a synthesis of compounds of
Formula I wherein R.sub.12 is --N(R.sub.4)(COR.sub.3) from
compounds of Formula 113.
[0145] Reaction Scheme 3 shows a synthesis of compounds of Formula
I wherein R.sub.12 is --N(R.sub.4)(SO.sub.2R.sub.3a) from compounds
of Formula 203.
[0146] Reaction Scheme 4 shows a synthesis of compounds of Formula
I wherein R.sub.12 is --N(R.sub.4)(CH.sub.2R.sub.3b) from compounds
of Formula 203.
[0147] Reaction Scheme 5 shows a synthesis of compounds of Formula
I wherein R.sub.12 is optionally substituted imidazolyl from
compounds of Formula 113.
[0148] Reaction Scheme 6 shows a synthesis of compounds of Formula
I wherein R.sub.12 is optionally substituted imidazolyl from
compounds of Formula 113.
[0149] Reaction Scheme 7 shows a synthesis of compounds of Formula
I wherein R.sub.12 is optionally substituted imidazolinyl from
compounds of Formula 113.
[0150] Reaction Scheme 8 shows a synthesis of compounds of Formula
I wherein R.sub.12 is optionally substituted imidazolinyl from
compounds of Formula 705.
[0151] Reaction Scheme 9 shows a synthesis of compounds of Formula
I wherein R.sub.12 is --N(R.sub.4)(COR.sub.3) and R.sub.3 is
--OR.sub.15, from compounds of Formula 203.
[0152] Reaction Scheme 10 shows a synthesis of compounds of Formula
I wherein R.sub.12 is --N(R.sub.4)(COR.sub.3) and R.sub.3 is
--NHR.sub.17, from compounds of Formula 203.
[0153] Reaction Scheme 11 shows a synthesis of compounds of Formula
I wherein R.sub.12 is optionally substituted imidazolyl.
[0154] Reaction Scheme 12 shows a synthesis of compounds of Formula
I wherein R.sub.12 is optionally substituted imidazolinyl from
compounds of Formula 113.
Starting Materials
[0155] The optionally substituted benzoic acids of Formula 101 and
the other reactants are commercially available, e.g., from Aldrich
Chemical Company, Milwaukee, Wis. or may be readily prepared by
those skilled in the art using commonly employed synthetic
methodology. ##STR5## Preparation of Compounds of Formula 103
[0156] Referring to Reaction Scheme 1, Step 1, an optionally
substituted carboxylic acid of Formula 101 is protected with a
suitable protecting group. For example, the following procedure is
used to prepare esters of the carboxylic acid. A solution of a
compound of Formula 101 and thionyl chloride in a polar aprotic
solvent (such as DMF) is warmed gently until the mixture becomes
homogeneous. The solution is concentrated. A lower alkanol, such as
methanol, is then added. The corresponding, optionally substituted
compound of Formula 103 is isolated and purified.
Preparation of Compounds of Formula 105
[0157] Referring to Reaction Scheme 1, Step 2, a compound of
Formula 103 is coupled to a compound of Formula 104. A variety of
reaction conditions can be used to effect this coupling, e.g.,
Sonogashira coupling conditions using a palladium catalyst such as
(diphenylphosphineferrocenyl)dichloropalladium or
tris(dibenzylidenacetone)-dipalladium; a base such as cesium
carbonate or triethylamine; and ligands such as triphenylarsine or
triphenylphosphine; Stille conditions using a palladium catalyst
such as tris(dibenzylidinacetone)-dipalladium; lithium chloride;
and triphenylarsine; or Suzuki coupling conditions wherein the
compound of Formula 104 is treated with a borane such as catechol
borane or 9-borabicyclo[3.3.1]nonane and the two fragments are
coupled using a palladium catalyst such as those described above, a
base such as cesium carbonate, and triphenylarsine.
[0158] According to one embodiment, a mixture of a compound of
Formula 103; a slight excess (preferably about 1.1 equivalents) of
an acetylenic compound of Formula 104; a palladium catalyst such as
dichlorobis(triphenylphosphine)palladium(II) and preferably about
0.025 equivalents of the catalyst; and cuprous iodide in a base
such as triethylamine is heated at about 50.degree. C. The
corresponding, optionally substituted compound of Formula 105 is
isolated and purified.
Preparation of Compounds of Formula 107
[0159] Referring to Reaction Scheme 1, Step 3, the protecting group
is then removed from the carboxylic acid. When the protecting group
is a lower alkyl ester, it can be removed by treatment with aqueous
alcoholic base at an elevated temperature. For example, methyl
esters can be converted to the corresponding carboxylic acid by
treatment of the ester with potassium hydroxide in a 1:1:1 solution
of methanol, THF, and alcohol at about 50.degree. C. for 30
minutes. The corresponding, optionally substituted compound of
Formula 107 is isolated and used without further purification.
Preparation of Compounds of Formula 109
[0160] Referring to Reaction Scheme 1, Step 4,
bis(acetonitrile)dichloropalladium (about 0.050 equivalents) is
added to a solution of a compound of Formula 107 and a base, such
as triethylamine, in a nonpolar, aprotic solvent such as THF. The
solution is maintained at an elevated temperature, preferably about
50.degree. C. The corresponding, optionally substituted
isochromen-1-one of Formula 109 is isolated and purified.
Preparation of Compounds of Formula 111
[0161] Referring to Reaction Scheme 1, Step 5, a solution of
isochromen-1-one of Formula 109 and an excess (preferably about 3
equivalents) of a primary amine of formula R.sub.1NH.sub.2 in a
nonpolar solvent such as toluene is heated at reflux, preferably
about 140.degree. C. The resulting amide is isolated and dissolved
in a polar, protic solvent such as methanol. Aqueous acid,
preferably about 5% aqueous hydrochloric acid is added to the
solution which is then heated to about 50.degree. C. Additional
aqueous alcholic acid may be added if required to complete the
reaction. The corresponding, optionally substituted
2H-isoquinolin-1-one of Formula 111 is isolated and purified.
Preparation of Compounds of Formula 113
[0162] Referring to Reaction Scheme 1, Step 6, the protecting group
is removed from the primary amine. In a preferred embodiment, the
protecting group is Boc and its removal can be accomplished through
treatment with aqueous TFA at room temperature. The product, a
compound of Formula 113 is isolated and can be used without further
purification.
[0163] In certain compounds of the invention, particular
stereoconfiguration can be preferred for the R.sub.2 substituent,
such as the (R) isomer, which can be obtained. An amine of Formula
113 is dissolved in an inert organic solvent (such as IPA) and
warmed to 60.degree. C. In a separate vessel, a resolving, agent
(such as dibenzoyl-D-tartaric acid) is dissolved, preferably in the
same warm solvent, and then quickly added (with agitation) to the
warm amine solution. The reaction mixture is left to crystallize by
cooling to room temperature over 16 hours under continuing
agitation. The desired isomer, e.g., the (R) isomer is isolated and
purified in the usual manner.
[0164] For the sake of brevity in the remaining description of the
synthesis of compounds of Formula I, it should be understood that
either single isomer or a mixture of isomers may be employed to
give the corresponding product. ##STR6## Preparation of Formula
203
[0165] Referring to Reaction Scheme 2, Step 1, to a solution of a
compound of Formula 113 is added successively a slight excess
(preferably about 1.2 equivalents) of an aldehyde comprising
R.sub.4' (i.e., a compound having the formula R.sub.4'CHO where
R.sub.4'CH.sub.2-- is equivalent to R.sub.4 and R.sub.4 is as
described above or is a protected precursor to such a substituent,
e.g., (3-oxo-propyl)-carbamic acid tert-butyl ester) and a reducing
agent such as sodium triacetoxyborohydride. The resulting mixture
is stirred for several hours. The product, a compound of Formula
203 is isolated and purified.
Preparation of Formula 205
[0166] Referring to Reaction Scheme 2, Step 2, to a solution of a
compound of Formula 203 and an amine base such as
diisopropylethylamine in a nonpolar, aprotic solvent such as
dichloromethane is added an R.sub.3 acyl chloride (such as
Cl--C(O)--R.sub.3 where R.sub.3 is as described above). The
resulting solution is stirred under nitrogen at room temperature
for several hours. The product, a compound of Formula 205 is
isolated and purified.
Preparation of Formula 207
[0167] Optionally, any protecting groups on compounds of Formula
205 are then removed. For example, if R.sub.4 comprises a protected
amine wherein the protecting group is a Boc group, the Boc group
can be removed by treatment of the compound of Formula 205 with an
acid such as trifluoroacetic acid in a nonpolar, aprotic solvent
such as dichloromethane, while maintaining the reaction at about
room temperature. The reaction is monitored e.g., by TLC. Upon
completion, the product, a compound of Formula 207 is isolated and
purified. ##STR7##
[0168] Referring to Reaction Scheme 3, to a solution of a compound
of Formula 203 and an amine base such as diisopropylethylamine in a
nonpolar, aprotic solvent such as dichloromethane is added a
compound having the formula Cl--S(O).sub.2--R.sub.3a or
O--(S(O).sub.2--R.sub.3a).sub.2 where R.sub.3a is as described
above. The resulting solution is stirred under nitrogen at room
temperature for several hours. The product, a compound of Formula
303 is isolated and purified. ##STR8##
[0169] Referring to Reaction Scheme 4, to a solution of a compound
of Formula 203 and an amine base such as diisopropylethylamine in a
nonpolar, aprotic solvent such as dichloromethane is added a
compound having the formula Cl--CH.sub.2--R.sub.3b where R.sub.3b
is as described above. The resulting solution is stirred under
nitrogen at room temperature or with heat for several hours. The
product, a compound of Formula 403 is isolated and purified.
##STR9## Preparation of Formula 503
[0170] Referring to Reaction Scheme 5, Step 1, to an optionally
substituted compound of Formula 113 dissolved in a polar, aprotic
solvent (such as DMF) in the presence of a base (such as potassium
carbonate) is added one equivalent of an optionally substituted
suitably protected aldehyde wherein such aldehyde further comprises
a leaving group, preferably, a halide. The solution is heated at
reflux, monitoring completion of the reaction (e.g., by TLC). The
reaction mixture is cooled and the corresponding, optionally
substituted compound of Formula 503 is isolated and purified.
Preparation of Formula 505
[0171] Referring to Reaction Scheme 5, Step 2, to an optionally
substituted compound of Formula 503 in an inert solvent (such as
dichloromethane) in the presence of about 1.5 molar equivalents of
an amine base (such as triethylamine) is added about 1.5 molar
equivalents of an R.sub.9 acid chloride, such as,
Cl--C(O)--R.sub.9, where R.sub.9 is as described above. The
reaction takes place, with stirring, at room temperature over a
period of 4 to 24 hours. Completion is monitored, e.g., by TLC. The
corresponding compound of Formula 505 is isolated and purified.
Preparation of Formula 507
[0172] Referring to Reaction Scheme 5, Step 3, a solution of a
compound of Formula 505 and an excess of ammonium acetate in acetic
acid is heated at reflux for 1-4 hours. Completion is monitored,
e.g., by TLC. The corresponding compound of Formula 507 is isolated
and purified. ##STR10## Preparation of Formula 603
[0173] Referring to Reaction Scheme 6, Step 1, a suspension of a
compound of Formula 113, an alpha-haloketone reagent of the Formula
R.sub.13'(CO)CH.sub.2X wherein X is a leaving group (such as a
halide) and R.sub.13' is as described herein, and about an
equivalent of a base, such as potassium carbonate in a polar,
aprotic solvent such as DMF is stirred at room temperature. The
reaction is diluted with water and the resulting solid, a compound
of Formula 603, is used in the subsequent step without further
purification.
Preparation of Formula 605
[0174] Referring to Reaction Scheme 6, Step 2, a solution of the
compound of Formula 603, about an equivalent of an amine base, such
as triethylamine and about an equivalent of an acid chloride (such
as a compound of Formula R.sub.9--COCl) in an organic solvent such
as methylene chloride is stirred at room temperature for several
hours. Completion is monitored, e.g., by TLC. The corresponding
compound of Formula 605 is isolated and purified.
Preparation of Formula 607
[0175] Referring to Reaction Scheme 6, Step 3, a solution of a
compound of Formula 605 and an excess of ammonium acetate in acetic
acid is heated at reflux using a Dean-Stark trap and condenser.
Completion is monitored, e.g., by TLC. The corresponding compound
of Formula 607 is isolated and purified.
Preparation of Formula 609
[0176] Optionally, in the event that group R.sub.13' comprises a
functionality bearing a protecting group, the protecting group is
removed. Thus, if R.sub.13' further comprises an amine bearing a
Pht group, the protecting group is removed as shown in Reaction
Scheme 6, Step 4. A solution of a compound of Formula 607 and an
excess of anhydrous hydrazine in a polar, protic solvent such as
ethanol is heated at reflux. The reaction is cooled to about
5.degree. C. and any precipitate is filtered off. The filtrate is
concentrated in vacuo and purified to yield a compound of Formula
609. ##STR11## Preparation of Formula 703
[0177] Referring to Reaction Scheme 7, Step 1, reductive amination
of amines of Formula 113 (prepared as described in WO 0130768) with
an optionally substituted, aldehyde-containing carbamic acid ester
(Seki et. al. Chem. Pharm. Bull. 1996, 44, 2061) gives urethane
intermediates. Removal of the Boc protecting group furnishes an
amine of Formula 705.
[0178] More specifically, to a solution of a compound of Formula
113 and an equivalent of a suitably protected aldehyde (Seki et.
al. Chem. Pharm. Bull. 1996, 44, 2061) in dichloromethane is added
a slight excess of a reducing agent, such as sodium
triacetoxyborohydride. The resultant cloudy mixture is maintained
at ambient temperature. Completion is monitored, e.g., by TLC. The
corresponding compound of Formula 703 is isolated and used in the
subsequent step without purification.
Preparation of Formula 705
[0179] Referring to Reaction Scheme 7, Step 2, the amine protecting
group, PG, is the removed. For example, when PG is Boc, to a
solution of a compound of Formula 703 in a nonpolar, aprotic
solvent such as dichloromethane is added a strong acid such as
trifluoroacetic acid. The resultant solution is maintained at
ambient temperature overnight and concentrated under reduced
pressure. The residue is isolated to give a compound of Formula 705
which was used in the subsequent step without purification.
Preparation of Formula 707
[0180] Referring to Reaction Scheme 7, Step 3, to a solution of a
compound of Formula 705 in a-nonpolar, aprotic solvent such as
dichloromethane is added an excess, preferably about two
equivalents of an amine base such as triethylamine, followed by
about an equivalent or slight excess of an acid chloride of the
formula R.sub.14COCl. The resultant solution is stirred at ambient
temperature for about 3 hours. Completion is monitored, e.g., by
TLC. The corresponding compound of Formula 707 is isolated and
purified.
Preparation of Formula 709
[0181] Referring, to Reaction Scheme 7, Step 4, a solution of a
compound of Formula 707 in an excess of phosphorus oxychloride is
heated at reflux. After 8 hours, the reaction mixture is allowed to
cool to ambient temperature and concentrated under reduced
pressure. The corresponding compound of Formula 709 is isolated and
purified. ##STR12## Preparation of Formula 709
[0182] As an alternative to Steps 3 and 4 of Reaction Scheme 7,
acylation of primary amines of Formula 705, followed by acetic acid
mediated cyclization, can proceed without isolation of the
intermediate amides to provide the target compound of Formula 709.
This is route is shown in Reaction Scheme 8.
[0183] More specifically, to a solution of a compound of Formula
705 in a nonpolar, aprotic solvent such as dichloromethane is added
an excess, preferably about two equivalents of an amine base, such
as triethylamine, followed by about an equivalent of an acid
chloride of formula R.sub.14COCl. The resultant solution is stirred
at ambient temperature for 2 hours, then evaporated under reduced
pressure. The resultant solid is treated with glacial acetic acid,
then the resultant suspension is heated at reflux for about 48
hours. The reaction is cooled to ambient temperature then
evaporated under reduced pressure. The corresponding compound of
Formula 709 is isolated and purified. ##STR13##
[0184] Referring to Reaction Scheme 9, a compound of Formula 203 is
reacted with a slight excess of a compound of the formula
R.sub.15O(CO)Cl in the presence of a base such as triethylamine in
a nonpolar, aprotic solvent such as dichloromethane. The product, a
compound of Formula 903 is isolated and purified. ##STR14##
[0185] Referring to Reaction Scheme 10, a compound of Formula 203
is treated with a slight excess of an isocyanate
R.sub.17--N.dbd.C.dbd.O in the presence of a base, such as
triethylamine, in a nonpolar, aprotic solvent, such as
dichloromethane. The product, a compound of Formula 1003, is
isolated and purified. ##STR15## Preparation of Compounds of
Formula 1103
[0186] Referring to Reaction Scheme 11, to a solution of a compound
of Formula 1101 in a nonpolar, aprotic solvent such as DMF are
added a base such as triethylamine and an excess (preferably, about
1.5 equivalents) of imidazole followed by about an equivalent of
tetrabutylammonium iodide. The resultant solution is heated to
about 90.degree. C., stirred for about 18 h and allowed to cool to
room temperature. The product, a compound of Formula 1103, is
isolated and purified. ##STR16## Preparation of Compounds of
Formula 1203
[0187] Referring to Reaction Scheme 12, Step 1, to a solution of a
compound of Formula 113 and an excess of an optionally substituted,
aldehyde-containing, carbamic acid ester such as
((S)-4-benzyloxycarbonylamino-1-formyl-butyl)-carbamic acid
tert-butyl ester) in a nonpolar, aprotic solvent such as
CH.sub.2Cl.sub.2 is added sodium triacetoxyborohydride. The mixture
is strirred overnight. The product, a compound of Formula 1203, is
isolated and purified.
Preparation of Compounds of Formula 1205
[0188] Referring to Reaction Scheme 12, Step 2, to a solution of a
compound of Formula 1203 in a nonpolar, aprotic solvent such as
toluene is added a base such as triethylamine followed by dropwise
addition of an excess of an acid chloride of the formula
R.sub.14--COCl. The reaction mixture is heated to about 80.degree.
C. for about 18 h, then at reflux for about 4 h. The product, a
compound of Formula 1205, is isolated and purified.
Preparation of Compounds of Formula 1207
[0189] Referring to Reaction Scheme 12, Step 3, a solution of a
compound of Formula 1205 in a solvent such as CH.sub.2Cl.sub.2/TFA
(preferably, about 4:1 CH.sub.2Cl.sub.2/TFA) is stirred at room
temperature. The reaction mixture is concentrated under reduced
pressure and the residue is diluted with a nonpolar, aprotic
solvent such as CH.sub.2Cl.sub.2 and washed with aqueous base. The
aqueous layer is extracted with a nonpolar, aprotic solvent such as
CH.sub.2Cl.sub.2 and the combined extracts are dried, filtered and
concentrated under reduced pressure. The residue is diluted with a
nonpolar, aprotic solvent such as THF and aqueous base (preferably,
saturated aqueous NaHCO.sub.3). The mixture is stirred at room
temperature for 10 days. The product, a compound of Formula 1207,
is isolated and purified.
Particular Processes and Last Steps
[0190] A compound of Formula 203: ##STR17## (where R.sub.4 is
optionally protected) is contacted with a slight molar excess of an
R.sub.3 chloride [such as, Cl--C(O)--R.sub.3,
Cl--S(O).sub.2--R.sub.3a, Cl--CH.sub.2--R.sub.3b,
Cl--C(O)--O--R.sub.15 and Cl--S(O).sub.2--NH--R.sub.3a] or an
isocyanate (such as O.dbd.C.dbd.N--R.sub.17) or an anhydride (such
as O[C(O)R.sub.15].sub.2 or O[S(O).sub.2R.sub.3a].sub.2) to give
the corresponding optionally protected compound of Formula I.
[0191] A compound of Formula 505, 605, 705, or 707 is optionally
cyclized by acid-mediated cyclization.
[0192] A racemic mixture of isomers of a compound of Formula I is
optionally placed on a chromatography column and separated into
(R)-- and (S)-enantiomers.
[0193] A compound of Formula I is optionally contacted with a
pharmaceutically acceptable acid or base to form the corresponding
acid or base addition salt.
[0194] A pharmaceutically acceptable acid addition salt of a
compound of Formula I is optionally contacted with a base to form
the corresponding free base of Formula I.
[0195] A pharmaceutically acceptable base addition salt of a
compound of Formula I is optionally contacted with an acid to form
tile corresponding free acid of Formula I.
Particular Embodiments of Compounds of the Invention
R.sub.1
[0196] When considering the compounds of Formula I, in one
embodiment, R.sub.1 is selected from hydrogen, optionally
substituted C.sub.1-C.sub.8 alkyl-, optionally substituted aryl-,
optionally substituted heteroaryl-, optionally substituted
aryl-C.sub.1-C.sub.4-alkyl-, and optionally substituted
heteroaryl-C.sub.1-C.sub.4-alkyl- (more particularly optionally
substituted aryl and optionally substituted
aryl-C.sub.1-C.sub.4-alkyl-). In a more particular embodiment
R.sub.1 is selected from hydrogen, optionally substituted
C.sub.1-C.sub.4 alkyl-, optionally substituted
phenyl-C.sub.1-C.sub.4-alkyl-, optionally substituted
naphthalenylmethyl-, optionally substituted phenyl-, and naphthyl-.
Even more particularly, R.sub.1 is optionally substituted
phenyl-C.sub.1-C.sub.4-alkyl-, optionally substituted
heteroaryl-C.sub.1-C.sub.4-alkyl-, or naphthalenylmethyl-.
[0197] Yet more particularly, R.sub.1 is naphthyl-, phenyl-,
bromophenyl-, chlorophenyl-, methoxyphenyl-, ethoxyphenyl-, tolyl-,
dimethylphenyl-, chorofluorophenyl-, methylchlorophenyl-,
ethylphenyl-, phenethyl-, benzyl-, chlorobenzyl-, methylbenzyl-,
methoxybenzyl-, cyanobenzyl-, hydroxybenzyl-, dichlorobenzyl-,
dimethoxybenzyl-, or naphthalenylmethyl-. More suitably, R.sub.1 is
benzyl-, cyanobenzyl-, methoxybenzyl-, or naphthalenylmethyl-. Most
particularly, R.sub.1 is benzyl-.
R.sub.2 and R.sub.2'
[0198] When considering the compounds of Formula I and as will be
appreciated by those skilled in the art, the compounds described
herein possess a potentially chiral center at the carbon to which
R.sub.2 and R.sub.2' are attached. The R.sub.2 and R.sub.2' groups
may be the same or different; if different, the compound is chiral
(i.e., has a stereogenic center). When R.sub.2 and R.sub.2' are
different, in particular embodiments R.sub.2' is hydrogen and
R.sub.2 is other than hydrogen. The invention contemplates the use
of pure enantiomers and mixtures of enantiomers, including racemic
mixtures, although the use of a substantially optically pure
enantiomer will generally be preferred. The term "substantially
pure" means having at least about 95% chemical purity with no
single impurity greater than about 1%. The term "substantially
optically pure" or "enantiomerically pure" means having at least
about 97.5% enantiomeric excess. In a a particular embodiment, the
stereogtenic center to which R.sub.2 and R.sub.2' are attached is
of the R configuration.
[0199] When considering the compounds of Formula I, in one
embodiment, R.sub.2 and R.sub.2' are independently chosen from
hydrogen, optionally substituted alkyl-, optionally substituted
alkoxy, optionally substituted aryl-, optionally substituted
aralkyl-, optionally substituted heteroaryl-, and optionally
substituted heteroaralkyl-.
[0200] In another embodiment, R.sub.2 and R.sub.2' taken together
form a 3- to 7-membered ring which may optionally be substituted
one or more of the following groups: hydroxyl, halogen
(particularly chloro and fluoro), optionally substituted
C.sub.1-C.sub.4 alkyl- (particularly methyl-), C.sub.1-C.sub.4
alkoxy (particularly methoxy), cyano, amino, substituted amino, or
carbamyl-.
[0201] In one embodiment, R.sub.2 is optionally substituted
C.sub.1-C.sub.4 alkyl-, and R.sub.2' is hydrogen or optionally
substituted C.sub.1-C.sub.4 alkyl-. More suitably, R.sub.2' is
hydrogen and R.sub.2 is optionally substituted C.sub.1-C.sub.4
alkyl-. In a most particular embodiment R.sub.2 is chosen from
methyl-, ethyl-, propyl (particularly, c-propyl or i-propyl), butyl
(particularly, t-butyl), methylthioethyl-, methylthiomethyl-,
aminobutyl-, (CBZ)aminobutyl-, cyclohexylmethyl-, benzyloxymethyl-,
methylsulfanylethyl-, methylsulfanylmethyl-, and hydroxymethyl-,
and R.sub.2' is hydrogen. Especially chosen embodiments are when
R.sub.2' is hydrogen and R.sub.2 is ethyl or propyl (particularly,
c-propyl or i-propyl). Even more particularly, R.sub.2 is i-propyl.
Yet more particularly, the stereogenic center to which R.sub.2 and
R.sub.2' is attached is of the R configuration.
[0202] In one embodiment, if either R.sub.2 or R.sub.2' is
hydrogen, then the other is not hydrogen. In another embodiment,
both R.sub.2 and R.sub.2' are hydrogen.
R.sub.5, R.sub.6, R.sub.7, and R.sub.8
[0203] In other embodiments R.sub.5, R.sub.6, R.sub.7, and R.sub.8
are independently chosen from hydrogen, hydroxyl, halogen
(particularly chloro and fluoro), optionally substituted
C.sub.1-C.sub.4 alkyl- (particularly methyl-), C.sub.1-C.sub.4
alkoxy (particularly methoxy), cyano, amino, substituted amino, or
carbamyl-. More particularly, R.sub.5, R.sub.6, R.sub.7, and
R.sub.8 are methoxy, methyl, trifluoromethyl, cyano, hydrogen or
halo. In particular embodiments, R.sub.5 is hydrogen or halo;
R.sub.6 is hydrogen, optionally substituted C.sub.1-C.sub.4 alkyl-
(particularly, methyl-) or halo; R.sub.7 is hydrogen, halo,
optionally substituted C.sub.1-C.sub.4 alkyl- (particularly,
methyl- or trifluoromethyl-), C.sub.1-C.sub.4 alkoxy (particularly,
methoxy), cyano, substituted amino, or carbamyl-; and R.sub.8 is
hydrogen, C.sub.1-C.sub.4 alkyl- (particularly, methyl-),
C.sub.1-C.sub.4 alkoxy (particularly, methoxy), hydroxy, or halo.
Still more particularly are the compounds where only one of
R.sub.5, R.sub.6, R.sub.7, and R.sub.8 is not hydrogen, especially
R.sub.7. Another particular embodiment is drawn to the compounds
where R.sub.7 and R.sub.8 are not hydrogen.
Compounds Wherein R.sub.12 is an Optionally Substituted
Imidazolyl
[0204] When R.sub.12 is an optionally substituted imidazolyl-, in
particular embodiments, R.sub.12 has the formula: ##STR18##
wherein
[0205] R.sub.9 is chosen from hydrogen, optionally substituted
C.sub.1-C.sub.8 alkyl-, optionally substituted aryl-, optionally
substituted aryl-C.sub.1-C.sub.4-alkyl-, optionally substituted
heteroaryl-C.sub.1-C.sub.4-alkyl-, optionally substituted
aryl-C.sub.1-C.sub.4-alkoxy, optionally substituted
heteroaryl-C.sub.1-C.sub.4-alkoxy, and optionally substituted
heteroaryl-; and
[0206] R.sub.13 and R.sub.13' are independently hydrogen,
optionally substituted C.sub.1-C.sub.8 alkyl-, optionally
substituted aryl-, or optionally substituted
aryl-C.sub.1-C.sub.4-alkyl-.
[0207] According to one embodiment, R.sub.9 is lower-alkyl; phenyl
substituted with optionally substituted C.sub.1-C.sub.4-alkyl-,
C.sub.1-C.sub.4-alkoxy-, and/or halo; phenyl-; benzyl-;
thiophenyl-; or thiophenyl- substituted with
C.sub.1-C.sub.4-alkyl-, C.sub.1-C.sub.4-alkoxy-, and/or halo. More
suitably, R.sub.9 is lower-alkyl; phenyl-; or phenyl- substituted
with one or more of the following groups: methyl, methoxy,
trifluoromethyl, or halo.
[0208] According to another embodiment, R.sub.13 is hydrogen and
R.sub.13' is substituted C.sub.1-C.sub.4 alkyl-. More suitably,
R.sub.13 is hydrogen and R.sub.13' is aminomethyl-, aminoethyl-,
aminopropyl-, acetylamino-methyl-, acetylaminoethyl-,
benzyloxycarbonylamino-methyl- or
benzyloxycarbonylamino-ethyl-.
Compounds Wherein R.sub.12 is an Optionally Substituted
Imidazolinyl
[0209] When R.sub.12 is an optionally substituted imidazolinyl, in
one embodiment, R.sub.12 has the formula ##STR19## wherein,
[0210] R.sub.14 is chosen from hydrogen, optionally substituted
C.sub.1-C.sub.8 alkyl-, optionally substituted aryl-, optionally
substituted aryl-C.sub.1-C.sub.4-alkyl-, optionally substituted
heteroaryl-, optionally substituted
heteroaryl-C.sub.1-C.sub.4-alkyl-; and
[0211] R.sub.10, R.sub.10', R.sub.11 and R.sub.11' are
independently chosen from hydrogen, optionally substituted
C.sub.1-C.sub.8 alkyl-, optionally substituted aryl-, and
optionally substituted aryl-C.sub.1-C.sub.4-alkyl-.
[0212] In one embodiment, R.sub.14 is lower-alkyl-;
methylenedioxyphenyl-; phenyl-; phenyl substituted with optionally
substituted C.sub.1-C.sub.4 alkyl-, C.sub.1-C.sub.4 alkoxy-, and/or
halo; benzyl-; thienyl substituted with C.sub.1-C.sub.4 alkyl;
benzyl; thiophenyl-; or thiophenyl- substituted with
C.sub.1-C.sub.4-alkyl-, C.sub.1-C.sub.4-alkoxy-, and/or halo. More
suitably, R.sub.14 is lower-alkyl; phenyl-; or phenyl- substituted
with one or more of the following groups: methyl, methoxy,
trifluoromethyl, or halo.
[0213] In one embodiment, R.sub.10, R.sub.10', R.sub.11', and
R.sub.11 are independently hydrogen or optionally substituted
C.sub.1-C.sub.4 alkyl-. More suitably, R.sub.11' it and R.sub.11
are hydrogen.
Compounds Wherein R.sub.12 is --NHR.sub.4, --NR.sub.4(COR.sub.3),
NR.sub.4(SO.sub.2R.sub.3a), or --NR.sub.4(CH.sub.2R.sub.3b)
R.sub.4
[0214] In one embodiment, R.sub.4 is chosen from hydrogen,
optionally substituted alkyl-, optionally substituted aryl-,
optionally substituted aralkyl-, optionally substituted
heteroaralkyl-, and optionally substituted heterocyclyl-. More
suitably, R.sub.4 is R.sub.16-alkylene-, and R.sub.16 is chosen
from alkoxy, amino, alkylamino, dialkylamino, carboxy, guanidine,
hydroxyl-, and N-heterocyclyl-.
[0215] In a more particular embodiment, R.sub.4 is selected from
optionally substituted lower-alkyl-, optionally substituted
cyclohexyl-; phenyl substituted with hydroxy, lower-alkoxy or
lower-alkyl-; benzyl-; heteroarylmethyl-; heteroarylethyl-; and
heteroarylpropyl-.
[0216] In a most particular embodiment, R.sub.4 is chosen from
methyl-, ethyl-, propyl-, butyl-, cyclohexyl-, carboxyethyl-,
carboxymethyl-, methoxyethyl-, hydroxyethyl-, hydroxypropyl-,
dimethylaminoethyl-, dimethylaminopropyl-, diethylaminoethyl-,
diethylaminopropyl-, aminopropyl-, methylaminopropyl-,
2,2-dimethyl-3-(dimethylamino)propyl-,
1-cyclohexyl-4-(diethylamino)butyl-, aminoethyl-, aminobutyl-,
aminopentyl-, aminohexyl-, aminoethoxyethyl-,
isopropylaminopropyl-, diisopropylaminoethyl-,
1-methyl-4-(diethylamino)butyl-, (t-Boc)aminopropyl-,
hydroxyphenyl-, benzyl-, methoxyphenyl-, methylmethoxyphenyl-,
dimethylphenyl-, tolyl-, ethylphenyl-, (oxopyrrolidinyl)propyl-,
(methoxycarbonyl)ethyl-, benzylpiperidinyl-, pyridinylethyl-,
pyridinylmethyl-, morpholinylethyl morpholinylpropyl-,
piperidinyl-, azetidinylmethyl-, azetidinylethyl-,
azetidinylpropyl-, pyrrolidinylmethyl-, pyrrolidinylethyl-,
pyrrolidinylpropyl-, piperidinylmethyl-, piperidinylethyl-,
imidazolylpropyl-, imidazolylethyl-, (ethylpyrrolidinyl)methyl-,
(methylpyrrolidinyl)ethyl-, (methylpiperidinyl)propyl-,
(methylpiperazinyl)propyl-, guanidino-methyl-, guanidino-ethyl-,
guanidino-propyl-, furanylmethyl and indolylethyl- (most especially
aminopropyl).
R.sub.3
[0217] When considering the compounds of Formula I, in a particular
embodiment R.sub.3 is selected from hydrogen, optionally
substituted alkyl-, optionally substituted aralkyl-, optionally
substituted heteroaralkyl-, optionally substituted heteroaryl-,
optionally substituted aryl-, R.sub.15O-- and R.sub.17--NH--,
wherein R.sub.15 is chosen from optionally substituted alkyl and
optionally substituted aryl and R.sub.17 is chosen from hydrogen,
optionally substituted alkyl and optionally substituted aryl-.
[0218] In a more particular embodiment, when R.sub.3 is not
R.sub.17NH-- or R.sub.15O--, R.sub.3 is chosen from optionally
substituted alkyl-; aryl- (including phenyl-, biphenyl-, and
naphthyl-); substituted aryl- (including phenyl substituted with
one or more cyano, halo, lower-alkyl-, lower-alkoxy,
hydroxy-loweralkyl-, nitro, carboxy, methylenedioxy,
trifluoromethoxy, or trifluoromethyl-); benzyl-; and optionally
substituted heteroaryl-.
[0219] In a most particular embodiment, when R.sub.3 is not
R.sub.17NH-- or R.sub.15O--, R.sub.3 is chosen from ethyl-,
propyl-, chloropropyl-, butoxy, heptyl-, butyl-, octyl-,
tridecanyl-, (ethoxycarbonyl)ethyl-, dimethylaminoethyl-,
dimethylaminomethyl-, phenyl-, naphthyl-, halophenyl-,
polyhalophenyl-, cyanophenyl-, hydroxymethylphenyl-,
halo(trifluoromethyl)phenyl-, chlorophenoxymethyl-, methoxyphenyl-,
carboxyphenyl-, ethylphenyl-, tolyl-,hydroxymethylphenyl-;
ethylphenyl-; biphenylyl-, methylenedioxyphenyl-,
methylsulfonylphenyl-, methoxychlorophenyl-, chloronaphthlyl-,
acetylphenyl-, methylhalophenyl-, trifluoromethylphenyl-,
trifluoromethoxyphenyl-, butylphenyl-, pentylphenyl-,
methylnitrophenyl-, phenoxymethyl-, dimethoxyphenyl-, phenylvinyl-,
nitrochlorophenyl-, nitrophenyl-, dinitrophenyl-,
bis(trifluoromethyl)phenyl-, benzyloxymethyl-, benzyl-, furanyl-,
benzofuranyl-, pyridinyl-, pyridyl-, indolyl-, methylpyridinyl-,
methylpyridyl-, (3-carbamoyl)pyridinyl-[nicotinamide],
3-carbamoyl-6-methylpyridinyl-, quinolinyl-, picolinyl-,
pyrazolyl-, pyrazinyl-, methylpyrazinyl-, morpholinomethyl-,
methylthiomethyl-, methoxymethyl-, imidazolyl-; isoxazolyl-,
methyl-isoxazolyl-; benzothiadiazolyl-; methylenedioxyphenyl-,
thienyl-, methylthienyl-, methyl-nicotinamidyl-; methyl-pyrazinyl;
benzodioxolyl; and methyl-thiophenyl-.
[0220] More suitably, R.sub.3 is tolyl-, halophenyl-,
halomethylphenyl-, hydroxymethylphenyl-, methylenedioxyphenyl-,
formylphenyl or cyanophenyl-.
[0221] In another particular embodiment, when R.sub.3 is
R.sub.17NH--, R.sub.17 is chosen from lower-alkyl-; cyclohexyl-;
phenyl-; and phenyl substituted with halo, lower-alkyl-,
loweralkoxy, or lower-alkylsulfanyl-.
[0222] In another particular embodiment, when R.sub.3 is
R.sub.17NH--, R.sub.17 is isopropyl-, butyl-, cyclohexyl-, phenyl-,
bromophenyl-, dichlorophenyl-, methoxyphenyl-, ethylphenyl-,
tolyl-, trifluoromethylphenyl or methylthiophenyl-.
[0223] In a particular embodiment, when R.sub.3 is R.sub.15O--,
R.sub.15 is chosen from lower-alkyl-; cyclohexyl-; phenyl-; and
phenyl substituted with halo, lower-alkyl-, loweralkoxy, or
lower-alkylsulfanyl-.
[0224] In a most particular embodiment, when R.sub.3 is
R.sub.15O--, R.sub.15 is isopropyl-, butyl-, cyclohexyl-, phenyl-,
bromophenyl-, dichlorophenyl-, methoxyphenyl-, ethylphenyl-,
tolyl-, trifluoromethylphenyl or methylthiophenyl-.
R.sub.3a
[0225] When R.sub.12 is --NR.sub.4(SO.sub.2R.sub.3a), R.sub.4 is as
described above and R.sub.3a is chosen from C.sub.1-C.sub.13
alkyl-; phenyl-; naphthyl-; phenyl substituted-with cyano, halo,
lower-alkyl-, lower-alkoxy, nitro, methylenedioxy, or
trifluoromethyl-; biphenylyl and heteroaryl-. More suitably,
R.sub.3a is chosen from phenyl substituted with halo, lower-alkyl-,
lower-alkoxy, cyano, nitro, methlenedioxy, or trifluoromethyl-; and
naphthyl-.
R.sub.3b
[0226] When R.sub.12 is --NR.sub.4(CH.sub.2R.sub.3b), R.sub.4 is as
described above and R.sub.3b is chosen from C.sub.1-C.sub.13
alkyl-; substituted lower-alkyl-; phenyl-; naphthyl-; phenyl
substituted with cyano, halo, lower-alkyl-, lower-alkoxy, nitro,
methylenedioxy, or trifluoromethyl-; biphenylyl-, benzyl and
heterocyclyl-. Most suitably, R.sub.3b is chosen from phenyl
substituted with one or more halo, methyl-, cyano,
trifluoromethyl-, trifluoromethoxy, carboxy, or methoxycarbonyl
groups; piperidinyl-; and naphthyl-. Even more suitably, R.sub.3b
is halophenyl-, methylhalophenyl-, polyhalophenyl-, tolyl-,
dimethylphenyl-, methoxyphenyl-, dimethoxyphenyl-, cyanophenyl-,
trifluoromethylphenyl-, trifluorometoxyphenyl-,
bis(trifluoromethyl)phenyl-, carboxyphenyl-, t-butylphenyl-,
methoxycarbonylphenyl-, piperidinyl-, and naphthyl-.
Particular Subgenus
[0227] In a particular subgenus of compounds of Formula I, [0228]
R.sub.1 is optionally substituted aryl-C.sub.1-C.sub.4-alkyl-,
optionally substituted heteroaryl-C.sub.1-C.sub.4-alkyl-, or
naphthalenylmethyl; [0229] R.sub.2 is optionally substituted
C.sub.1-C.sub.4-alkyl-; [0230] R.sub.2 is hydrogen; [0231] R.sub.2'
is hydrogen, methyl, or cyano; [0232] R.sub.5, R.sub.6, and R.sub.8
are hydrogen; and [0233] R.sub.12 is optionally substituted
imidazolyl-, optionally substituted imidazolinyl-, --NHR.sub.4;
--N(R.sub.4)(COR.sub.3); --N(R.sub.4)(SO.sub.2R.sub.3a); or
--N(R.sub.4)(CH.sub.2R.sub.3b).
[0234] In a particular subgenus of compounds of Formula I wherein
R.sub.12 is optionally substituted imidazolyl-, [0235] R.sub.1,
R.sub.2, R.sub.2', R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are as
defined above; [0236] R.sub.9 is lower-alkyl; phenyl-; or phenyl-
substituted with one or more of the following groups: methyl,
methoxy, trifluoromethyl, or halo; and [0237] R.sub.13 is hydrogen
and R.sub.13' is substituted C.sub.1-C.sub.4 alkyl- (especially,
aminomethyl-, aminoethyl-, aminopropyl-, acetylamino-methyl-,
acetylaminoethyl-, benzyloxycarbonylamino-methyl- or
benzyloxycarbonylamino-ethyl-.)
[0238] In a particular subgenus of compounds of Formula I wherein
R.sub.12 is optionally substituted imidazolinyl-, [0239] R.sub.1,
R.sub.2, R.sub.2', R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are as
defined above; [0240] R.sub.14 is lower-alkyl; phenyl-; or phenyl-
substituted with one or more of the following groups: methyl,
methoxy, trifluoromethyl, or halo; and [0241] R.sub.10, R.sub.10',
R.sub.11', and R.sub.11 are independently hydrogen or optionally
substituted C.sub.1-C.sub.4 alkyl-. More particularly, R.sub.11'
and R.sub.11 are hydrogen.
[0242] In a particular subgenus of compounds of Formula I wherein
R.sub.12 is --NHR.sub.4. R.sub.1, R.sub.2, R.sub.2', R.sub.5,
R.sub.6, R.sub.7, and R.sub.8 are as defined above; [0243] R.sub.4
is chosen from hydrogen, optionally substituted alkyl-, optionally
substituted aryl-, optionally substituted aralkyl-, optionally
substituted heteroaralkyl-, and optionally substituted
heterocyclyl-.
[0244] In a particular subgenus of compounds of Formula I wherein
R.sub.12 is --NR.sub.4(COR.sub.3). [0245] R.sub.1, R.sub.2,
R.sub.2', R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are as defined
above; [0246] R.sub.4 is chosen from hydrogen, optionally
substituted alkyl-, optionally substituted aryl-, optionally
substituted aralkyl-, optionally substituted heteroaralkyl-, and
optionally substituted heterocyclyl- and [0247] R.sub.3 is selected
from hydrogen, optionally substituted alkyl-, optionally
substituted aralkyl-, optionally substituted heteroaralkyl-,
optionally substituted heteroaryl-, optionally substituted aryl-,
R.sub.15O-- and R.sub.17--NH--, wherein R.sub.15 is chosen from
optionally substituted alkyl and optionally substituted aryl and
R.sub.17 is chosen from hydrogen, optionally substituted alkyl and
optionally substituted aryl.
[0248] More particularly, R.sub.1, R.sub.2, R.sub.2', R.sub.5,
R.sub.6, R.sub.7, and R.sub.8 are as defined above; [0249] R.sub.4
is selected from optionally substituted lower-alkyl-, optionally
substituted cyclohexyl-; phenyl substituted with hydroxy,
lower-alkoxy, or lower-alkyl-; benzyl-; heteroarylmethyl-;
heteroarylethyl-; heteroarylpropyl- (especially R.sub.4 is
R.sub.16-alkylene-, and R.sub.16 is chosen from alkoxy, amino,
alkylamino, dialkylamino, carboxy, hydroxyl-, and N-heterocyclyl-);
and [0250] R.sub.3 is selected from hydrogen, optionally
substituted alkyl-, optionally substituted aralkyl-, optionally
substituted heteroaralkyl-, optionally substituted heteroaryl-,
optionally substituted aryl-, R.sub.15O-- and R.sub.17--NH--,
wherein R.sub.15 is chosen from optionally substituted alkyl and
optionally substituted aryl and R.sub.17 is chosen from hydrogen,
optionally substituted alkyl and optionally substituted aryl.
[0251] In a particular subgenus of compounds of Formula I wherein
R.sub.12 is --NR.sub.4(COR.sub.3). [0252] R.sub.1, R.sub.2,
R.sub.2', R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are as defined
above; [0253] R.sub.4 is chosen from hydrogen, optionally
substituted alkyl-, optionally substituted aryl-, optionally
substituted aralkyl-, optionally substituted heteroaralkyl-, and
optionally substituted heterocyclyl- and and [0254] R.sub.3 is
selected from optionally substituted alkyl-; aryl-; substituted
aryl-; benzyl-; and optionally substituted heteroaryl-.
[0255] In a particular subgenus of compounds of Formula I wherein
R.sub.12 is --NR.sub.4(COR.sub.3), [0256] R.sub.1, R.sub.2,
R.sub.2', R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are as defined
above; [0257] R.sub.3 is tolyl-, halophenyl-, halomethylphenyl-,
hydroxymethylphenyl-, methylenedioxyphenyl-, formylphenyl or
cyanophenyl-; and [0258] R.sub.4 is optionally substituted
lower-alkyl-, optionally substituted cyclohexyl-; phenyl
substituted with hydroxy, lower-alkoxy or lower-alkyl-; benzyl-;
heteroarylmethyl-; heteroarylethyl-; or heteroarylpropyl-
(especially R.sub.4 is R.sub.16-alkylene- and R.sub.16 is alkoxy,
amino, alkylamino, dialkylamino, carboxy, guanidine, hydroxyl, or
N-heterocyclyl-).
[0259] In a particular subgenus of compounds of Formula I wherein
R.sub.12 is --N(R.sub.4)(CH.sub.2R.sub.3b), [0260] R.sub.1,
R.sub.2, R.sub.2', R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are as
defined above; [0261] R.sub.4 is chosen from hydrogen, optionally
substituted alkyl-, optionally substituted aryl-, optionally
substituted aralkyl-, optionally substituted heteroaralkyl-, and
optionally substituted heterocyclyl- and [0262] R.sub.3b is chosen
from phenyl substituted with one or more halo, methyl-, cyano,
trifluoromethyl-, trifluoromethoxy, carboxy, or methoxycarbonyl
groups; piperidinyl-; and naphthyl-.
[0263] In a particular subgenus of compounds of Formula I wherein
R.sub.12 is --NR.sub.4(SO.sub.2R.sub.3a), [0264] R.sub.1, R.sub.2,
R.sub.2', R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are as defined
above; [0265] R.sub.4 is chosen from hydrogen, optionally
substituted alkyl-, optionally substituted aryl-, optionally
substituted aralkyl-, optionally substituted heteroaralkyl-, and
optionally substituted heterocyclyl- and [0266] R.sub.3a is chosen
from phenyl substituted with halo, lower-alkyl-, lower-alkoxy,
cyano, nitro, methylenedioxy, or trifluoromethyl-; and
naphthyl-.
[0267] Particular compounds include: [0268]
N-(3-Amino-propyl)-N-[1-(2-benzyl-4-methoxy-1-oxo-1,2-dihydro-isoquinolin-
-3-yl)-2-methyl-propyl]-4-methyl-benzamide; [0269]
N-(3-Amino-propyl)-N-[1-(2-benzyl-4-methoxy-1-oxo-1,2-dihydro-isoquinolin-
-3-yl)-2-methyl-propyl]-2-methoxy-acetamide; [0270]
3-(1-Amino-2-methyl-propyl)-2-benzyl-6-chloro-2H-isoquinolin-1-one;
[0271]
N-(3-Amino-propyl)-N-[1-(2-benzyl-6-chloro-1-oxo-1,2-dihydro-isoq-
uinolin-3-yl)-2-methyl-propyl]-4-methyl-benzamide; [0272]
N-(3-Amino-propyl)-N-[1-(2-benzyl-6-chloro-1-oxo-1,2-dihydro-isoquinolin--
3-yl)-2-methyl-propyl]-2-methoxy-acetamide; [0273]
N-(3-Amino-propyl)-N-[1-(2-benzyl-6-chloro-1-oxo-1,2-dihydro-isoquinolin--
3-yl)-2-methyl-propyl]-4-bromo-benzamide; [0274]
2-Benzyl-6-chloro-3-[1-(4,4-dimethyl-2-p-tolyl-4,5-dihydro-imidazol-1-yl)-
-2-methyl-propyl]-2H-isoquinolin-1-one; [0275]
2-Benzyl-6-chloro-3-{1-[2-(3,4-dimethoxy-phenyl)-4,4-dimethyl-4,5-dihydro-
-imidazol-1-yl]-2-methyl-propyl}-2H-isoquinolin-1-one; [0276]
2-Benzyl-6-chloro-3-{1-[2-(3,4-dichloro-phenyl)-4,4-dimethyl-4,5-dihydro--
imidazol-1-yl]-2-methyl-propyl}-2H-isoquinolin-1-one; [0277]
2-Benzyl-6-chloro-3-[1-(4,4-dimethyl-2-m-tolyl-4,5-dihydro-imidazol-1-yl)-
-2-methyl-propyl]-2H-isoquinolin-l -one; [0278]
2-Benzyl-6-chloro-3-{1-[4,4-dimethyl-2-(3-trifluoromethyl-phenyl)-4,5-dih-
ydro-imidazol-1-yl]-2-methyl-propyl}-2H-isoquinolin-1-one; [0279]
2-Benzyl-6-chloro-3-{1-[2-(3-fluoro-phenyl)-4,4-dimethyl-4,5-dihydro-imid-
azol-1-yl]-2-methyl-propyl}-2H-isoquinolin-1-one; [0280]
2-Benzyl-6-chloro-3-{1-[2-(3-fluoro-4-methyl-phenyl)-4,4-dimethyl-4,5-dih-
ydro-imidazol-1-yl]-2-methyl-propyl}-2H-isoquinolin-1-one; [0281]
2-Benzyl-6-chloro-3-[1-(2-isopropyl-4,4-dimethyl-4,5-dihydro-imidazol-1-y-
l)-2-methyl-propyl]-2H-isoquinolin-1-one; [0282]
2-Benzyl-6-chloro-3-{1-[2-(4-methoxy-phenyl)-4,4-dimethyl-4,5-dihydro-imi-
dazol-1-yl]-2-methyl-propyl}-2H-isoquinolin-1-one; and [0283]
2-Benzyl-3-{1-[2-(4-bromo-phenyl)-4,4-dimethyl-4,5-dihydro-imidazol-1-yl]-
-2-methyl-propyl}-6-chloro-2H-isoquinolin-1-one.
Utility, Testing and Administration
[0283] General Utility
[0284] Once made, the compounds of the invention find use in a
variety of applications involving alteration of mitosis. As will be
appreciated by those skilled in the art, mitosis may be altered in
a variety of ways; that is, one can affect mitosis either by
increasing or decreasing the activity of a component in the mitotic
pathway. Stated differently, mitosis may be affected (e.g.,
disrupted) by disturbing equilibrium, either by inhibiting or
activating certain components. Similar approaches may be used to
alter meiosis.
[0285] In a particular embodiment, the compounds of the invention
are used to inhibit mitotic spindle formation, thus causing
prolonged cell cycle arrest in mitosis. By "inhibit" in this
context is meant decreasing or interfering with mitotic spindle
formation or causing mitotic spindle dysfunction. By "mitotic
spindle formation" herein is meant organization of microtubules
into bipolar structures by mitotic kinesins. By "mitotic spindle
dysfunction" herein is meant mitotic arrest and monopolar spindle
formation.
[0286] The compounds of the invention are useful to bind to, and/or
inhibit the activity of, a mitotic kinesin, KSP. In one embodiment,
the KSP is human KSP, although the compounds may be used to bind to
or inhibit the activity of KSP kinesins from other organisms. In
this context, "inhibit" means either increasing or decreasing
spindle pole separation, causing malformation, i.e., splaying, of
mitotic spindle poles, or otherwise causing morphological
perturbation of the mitotic spindle. Also included within the
definition of KSP for these purposes are variants and/or fragments
of KSP. See U.S. Pat. No. 6,437,115, hereby incorporated by
reference in its entirety. The compounds of the invention have been
shown to have specificity for KSP. However, the present invention
includes the use of the compounds to bind to or modulate other
mitotic kinesins.
[0287] The compounds of the invention are used to treat cellular
proliferation diseases. Such disease states which can be treated by
the compounds, compositions and methods provided herein include,
but are not limited to, cancer (further discussed below),
autoimmune disease, fungal disorders, arthritis, graft rejection,
inflammatory bowel disease, cellular proliferation induced after
medical procedures, including, but not limited to, surgery,
angioplasty, and the like. Treatment includes inhibiting cellular
proliferation. It is appreciated that in some cases the cells may
not be in an abnormal state and still require treatment. Thus, in
one embodiment, the invention herein includes application to cells
or individuals afflicted or subject to impending affliction with
any one of these disorders or states.
[0288] The compounds, compositions and methods provided herein are
particularly deemed useful for the treatment of cancer including
solid tumors such as skin, breast, brain, cervical carcinomas,
testicular carcinomas, etc. More particularly, cancers that may be
treated by the compounds, compositions and methods of the invention
include, but are not limited to: Cardiac: sarcoma (angiosarcoma,
fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma; rhabdomyoma,
fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma
(squamous cell, undifferentiated small cell, undifferentiated large
cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial
adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;
Gastrointestinal: esophagus (squamous cell carcinoma,
adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma,
lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma,
insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma),
small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's
sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma),
large bowel (adenocarcinoma, tubular adenoma, villous adenoma,
hamartoma, leiomyoma); Genitourinary tract: kidney (adenocarcinoma,
Wilm's tumor (nephroblastoma), lymphoma, leukemia), bladder and
urethra (squamous cell carcinoma, transitional cell carcinoma,
adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis
(seminoma, teratoma, embryonal carcinoma, teratocarcinoma,
choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,
fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma
(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,
angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic
sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous
histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma
(reticulum cell sarcoma), multiple myeloma, malignant giant cell
tumor chordoma, osteochronfroma (osteocartilaginous exostoses),
benign chondroma, chondroblastoma, chondromyxofibroma, osteoid
osteoma and giant cell tumors; Nervous system: skull (osteoma,
hemangioma, granuloma, xanthoma, osteitis deformans), meninges
(meningioma, meninoiosarcoma, gliomatosis), brain (astrocytonia,
medulloblastoma, glioma, ependymoma, germinoma (pinealoma),
glioblastonma multiform, oligodendroglioma, schwannoma,
retinoblastoma, congenital tumors), spinal cord neurofibroma,
meningioma, glioma, sarcoma); Gynecological: uterus (endometrial
carcinoma), cervix (cervical carcinoma, pre-tumor cervical
dysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma,
mucinous cystadenocarcinoma, unclassified carcinoma),
granulosa-thecal cell tumors, Sertoli-Leydig, cell tumors,
dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,
intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),
vagina (clear cell carcinoma, squamous cell carcinoma, botryoid
sarcoma (embryonal rhabdomyosarconma), fallopian tubes (carcinoma);
Hematologic: blood (myeloid leukemia (acute and chronic), acute
lymphoblastic leukemia, chronic lymphocytic leukemia,
myeloproliferative diseases, multiple myeloma, myelodysplastic
syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant
lymphoma); Skin: malignant melanoma, basal cell carcinoma, squamous
cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma,
angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands:
neuroblastoma. Thus, the term "cancerous cell" as provided herein,
includes a cell afflicted by any one of the above identified
conditions.
Testing
[0289] For assay of KSP-modulating activity, generally either KSP
or a compound according to the invention is non-diffusably bound to
an insoluble support having isolated sample receiving areas (e.g.,
a microtiter plate, an array, etc.). The insoluble support may be
made of any material to which the sample can be bound, is readily
separated from soluble material, and is otherwise compatible with
the overall method of screening. The surface of such supports may
be solid or porous and of any convenient shape. Examples of
suitable insoluble supports include microtiter plates, arrays,
membranes and beads. These are typically made of glass, plastic
(e.g., polystyrene), polysaccharides, nylon or nitrocellulose,
Teflon.TM., etc. Microtiter plates and arrays are especially
convenient because a large number of assays can be carried out
simultaneously, using small amounts of reagents and samples. The
particular manner of binding of the sample is not crucial so long
as it is compatible with the reagents and overall methods of the
invention, maintains the activity of the sample and is
nondiffusable. Particular methods of binding include the use of
antibodies (which do not sterically block either the ligand binding
site or activation sequence when the protein is bound to the
support), direct binding to "sticky" or ionic supports, chemical
crosslinking, the synthesis of the protein or agent on the surface,
etc. Following binding of the sample, excess unbound material is
removed by washing. The sample receiving areas may then be blocked
through incubation with bovine serum albumin (BSA), casein or other
innocuous protein or other moiety.
[0290] The compounds of the invention may be used on their own to
inhibit the activity of a mitotic kinesin, particularly KSP. In one
embodiment, a compound of the invention is combined with KSP and
the activity of KSP is assayed. Kinesin (including KSP) activity is
known in the art and includes one or more kinesin activities.
Kinesin activities include the ability to affect ATP hydrolysis;
microtubule binding; gliding and polymerization/depolymerization
(effects on microtubule dynamics); binding to other proteins of the
spindle; binding to proteins involved in cell-cycle control;
serving as a substrate to other enzymes, such as kinases or
proteases; and specific kinesin cellular activities such as spindle
pole separation.
[0291] Methods of performing motility assays are well known to
those of skill in the art. (See e.g., Hall, et al. (1996), Biophys.
J., 71: 3467-3476, Turner et al., 1996, AnaL Biochem. 242 (1):20-5;
Gittes et al., 1996, Biophys. J. 70(1): 418-29; Shirakawa et al.,
1995, J. Exp. BioL 198: 1809-15; Winkelmann et al., 1995, Biophys.
J. 68: 2444-53; Winkelmann et al., 1995, Biophys. J. 68: 72S.)
[0292] Methods known in the art for determining ATPase hydrolysis
activity also can be used. Suitably, solution based assays are
utilized. U.S. Pat. No. 6,410,254, hereby incorporated by reference
in its entirety, describes such assays. Alternatively, conventional
methods are used. For example, P.sub.i release from kinesin can be
quantified. In one embodiment, the ATPase hydrolysis activity assay
utilizes 0.3 M PCA (perchloric acid) and malachite green reagent
(8.27 mM sodium molybdate II, 0.33 mM malachite green oxalate, and
0.8 mM Triton X-100). To perform the assay, 10 .mu.L of the
reaction mixture is quenched in 90 .mu.L of cold 0.3 M PCA.
Phosphate standards are used so data can be converted to mM
inorganic phosphate released. When all reactions and standards have
been quenched in PCA, 100 .mu.L of malachite green reagent is added
to the relevant wells in e.g., a microtiter plate. The mixture is
developed for 10-15 minutes and the plate is read at an absorbance
of 650 nm. If phosphate standards were used, absorbance readings
can be converted to mM P.sub.i and plotted over time. Additionally,
ATPase assays known in the art include the luciferase assay.
[0293] ATPase activity of kinesin motor domains also can be used to
monitor the effects of agents and are well known to those skilled
in the art. In one embodiment ATPase assays of kinesin are
performed in the absence of microtubules. In another embodiment,
the ATPase assays are performed in the presence of microtubules.
Different types of agents can be detected in the above assays. In a
one embodiment, the effect of an agent is independent of the
concentration of microtubules and ATP. In another embodiment, the
effect of the agents on kinesin ATPase can be decreased by
increasing the concentrations of ATP, microtubules or both. In yet
another embodiment, the effect of the agent is increased by
increasing concentrations of ATP, microtubules or both.
[0294] Compounds that inhibit the biochemical activity of KSP in
vitro may then be screened in vivo. In vivo screening methods
include assays of cell cycle distribution, cell viability, or the
presence, morphology, activity, distribution, or number of mitotic
spindles. Methods for monitoring cell cycle distribution of a cell
population, for example, by flow cytometry, are well known to those
skilled in the art, as are methods for determining cell viability.
See for example, U.S. Pat. No. 6,437,115, hereby incorporated by
reference in its entirety. Microscopic methods for monitoring
spindle formation and malformation are well known to those of skill
in the art (see, e.g., Whitehead and Rattner (1998), J. Cell Sci.
111:2551-61; Galgio et al, (1996) J. Cell Biol., 135:399-414), each
incorporated herein by reference in its entirety.
[0295] The compounds of the invention inhibit the KSP kinesin. One
measure of inhibition is IC.sub.50, defined as the concentration of
the compound at which the activity of KSP is decreased by fifty
percent relative to a control. Preferred compounds have IC.sub.50's
of less than about 1 mM, with preferred embodiments having
IC.sub.50's of less than about 100 .mu.M, with more preferred
embodiments having, IC.sub.50's of less than about 10 .mu.M, with
particularly preferred embodiments having IC.sub.50's of less than
about 1 .mu.M, and especially preferred embodiments having
IC.sub.50's of less than about 100 nM, and with the most preferred
embodiments having IC.sub.50's of less than about 10 nM.
Measurement of IC.sub.50 is done using an ATPase assay such as
described herein.
[0296] Another measure of inhibition is K.sub.i. For compounds with
IC.sub.50's less than 1 .mu.M, the K.sub.i or K.sub.d is defined as
the dissociation rate constant for the interaction of the compounds
described herein with KSP. Preferred compounds have K.sub.i's of
less than about 100 .mu.M, with preferred embodiments having
K.sub.i's of less than about 10 .mu.M, and particularly preferred
embodiments having K.sub.i's of less than about 1 .mu.M and
especially preferred embodiments having K.sub.i's of less than
about 100 nM, and with the most preferred embodiments having
K.sub.i's of less than about 10 nM.
[0297] The K.sub.i for a compound is determined from the IC.sub.50
based on three assumptions and the Michaelis-Menten equation.
First, only one compound molecule binds to the enzyme and there is
no cooperativity. Second, the concentrations of active enzyme and
the compound tested are known (i.e., there are no significant
amounts of impurities or inactive forms in the preparations).
Third, the enzymatic rate of the enzyme-inhibitor complex is zero.
The rate (i.e., compound concentration) data are fitted to the
equation: V = V max .times. E 0 [ I - ( E 0 + I 0 + Kd ) - ( E 0 +
I 0 + Kd ) 2 - 4 .times. E 0 .times. I 0 2 .times. E 0 ] ##EQU1##
where V is the observed rate, V.sub.max is the rate of the free
enzyme, I.sub.0 is the inhibitor concentration, E.sub.0 is the
enzyme concentration, and K.sub.d is the dissociation constant of
the enzyme-inhibitor complex.
[0298] Another measure of inhibition is GI.sub.50, defined as the
concentration of the compound that results in a decrease in the
rate of cell growth by fifty percent. Preferred compounds have
GI.sub.50's of less than about 1 mM; those having a GI.sub.50 of
less than about 20 .mu.M are more preferred; those having a
GI.sub.50 of less than about 10 .mu.M more so; those having a
GI.sub.50 of less than about 1 .mu.M more so; those having a
GI.sub.50 of less than about 100 nM more so; and those having a
GI.sub.50 of less than about 10 nM even more so. Measurement of
GI.sub.50 is done using a cell proliferation assay such as
described herein. Compounds of this class were found to inhibit
cell proliferation.
[0299] In vitro potency of small molecule inhibitors is determined,
for example, by assaying human ovarian cancer cells (SKOV3) for
viability following a 72-hour exposure to a 9-point dilution series
of compound. Cell viability is determined by measuring the
absorbance of formazon, a product formed by the bioreduction of
MTS/PMS, a commercially available reagent. Each point on the
dose-response curve is calculated as a percent of untreated control
cells at 72 hours minus background absorption (complete cell
kill).
[0300] Anti-proliferative compounds that have been successfully
applied in the clinic to treatment of cancer (cancer
chemotherapeutics) have GI.sub.50's that vary greatly. For example,
in A549 cells, paclitaxel GI.sub.50 is 4 nM, doxorubicin is 63 nM,
5-fluorouracil is 1 .mu.M, and hydroxyurea is 500 .mu.M (data
provided by National Cancer Institute, Developmental Therapeutic
Program, http://dtp.nci.nih.gov/). Therefore, compounds that
inhibit cellular proliferation, irrespective of the concentration
demonstrating inhibition, have potential clinical usefulness.
[0301] To employ the compounds of the invention in a method of
screening for compounds that bind to [(SP kinesin, the KSP is bound
to a support, and a compound of the invention is added to the
assay. Alternatively, the compound of the invention is bound to the
support and KSP is added. Classes of compounds among which novel
binding agents may be sought include specific antibodies,
non-natural binding agents identified in screens of chemical
libraries, peptide analogs, etc. Of particular interest are
screening assays for candidate agents that have a low toxicity for
human cells. A wide variety of assays may be used for this purpose,
including labeled in vitro protein-protein binding assays,
electrophoretic mobility shift assays, immunoassays for protein
binding, functional assays (phosphorylation assays, etc.) and the
like.
[0302] The determination of the binding of the compound of the
invention to KSP may be done in a number of ways. In one
embodiment, tile compound is labeled, for example, with a
fluorescent or radioactive moiety, and binding is determined
directly. For example, this may be done by attaching all or a
portion of KSP to a solid support, adding a labeled test compound
(for example a compound of the invention in which at least one atom
has been replaced by a detectable isotope), washing off excess
reagent, and determining whether the amount of the label is that
present on the solid support.
[0303] By "labeled" herein is meant that the compound is either
directly or indirectly labeled with a label which provides a
detectable signal, e.g., radioisotope, fluorescent tag, enzyme,
antibodies, particles such as magnetic particles, chemiluminescent
tag, or specific binding molecules, etc. Specific binding molecules
include pairs, such as biotin and streptavidin, digoxin and
antidigoxin etc. For the specific binding members, the
complementary member would normally be labeled with a molecule
which provides for detection, in accordance with known procedures,
as outlined above. The label can directly or indirectly provide a
detectable signal.
[0304] In some embodiments, only one of the components is labeled.
For example, the kinesin proteins may be labeled at tyrosine
positions using .sup.125I, or with fluorophores. Alternatively,
more than one component may be labeled with different labels; using
.sup.125I for the proteins, for example, and a fluorophor for the
antimitotic agents.
[0305] The compounds of the invention may also be used as
competitors to screen for additional drug candidates. "Candidate
agent" or "drug candidate" or grammatical equivalents as used
herein describe any molecule, e.g., protein, oligopeptide, small
organic molecule, polysaccharide, polynucleotide, etc., to be
tested for bioactivity. They may be capable of directly or
indirectly altering the cellular proliferation phenotype or the
expression of a cellular proliferation sequence, including both
nucleic acid sequences and protein sequences. In other cases,
alteration of cellular proliferation protein binding and/or
activity is screened. Screens of this sort may be performed either
in the presence or absence of microtubules. In the case where
protein binding or activity is screened, particular embodiments
exclude molecules already known to bind to that particular protein,
for example, polymer structures such as microtubules, and energy
sources such as ATP. Particular embodiments of assays herein
include candidate agents which do not bind the cellular
proliferation protein in its endogenous native state termed herein
as "exogenous" agents. In another embodiment, exogenous agents
further exclude antibodies to KSP.
[0306] Candidate agents can encompass numerous chemical classes,
though typically they are organic molecules, preferably they are
small organic compounds having a molecular weight of more than 100
and less than about 2,500 daltons. Candidate agents comprise
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding and lipophilic binding, and
typically include at least an amine, carbonyl-, hydroxyl-, ether,
or carboxyl group, preferably at least two of the functional
chemical groups. The candidate agents often comprise cyclical
carbon or heterocyclic structures and/or aromatic or polyaromatic
structures substituted with one or more of the above functional
groups. Candidate agents are also found among biomolecules
including peptides, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives, structural analogs or combinations
thereof.
[0307] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, and/or amidification to produce
structural analogs.
[0308] Competitive screening assays may be done by combining KSP
and a drug candidate in a first sample. A second sample comprises a
compound of the present invention, KSP and a drug candidate. This
may be performed in either the presence or absence of microtubules.
The binding of the drug candidate is determined for both samples,
and a change, or difference in binding between the two samples
indicates the presence of a drug candidate capable of binding to
KSP and potentially inhibiting its activity. That is, if the
binding of the drug candidate is different in the second sample
relative to the first sample, the drug candidate is capable of
binding to KSP.
[0309] In a particular embodiment, the binding of the candidate
agent to KSP is determined through the use of competitive binding
assays. In this embodiment, the competitor is a binding moiety
known to bind to KSP, such as an antibody, peptide, binding
partner, ligand, etc. Under certain circumstances, there may be
competitive binding as between the candidate agent and the binding
moiety, with the binding moiety displacing the candidate agent.
[0310] In one embodiment, the candidate agent is labeled. Either
the candidate agent, or the competitor, or both, is added first to
KSP for a time sufficient to allow binding, if present. Incubations
may be performed at-any temperature which facilitates optimal
activity, typically between 4 and 40.degree. C.
[0311] Incubation periods are selected for optimum activity, but
may also be optimized to facilitate rapid high throughput
screening. Typically between 0.1 and 1 hour will be sufficient.
Excess reagent is generally removed or washed away. The second
component is then added, and the presence or absence of the labeled
component is followed, to indicate binding.
[0312] In another embodiment, the competitor is added first,
followed by the candidate agent. Displacement of the competitor is
an indication the candidate agent is binding to KSP and thus is
capable of binding to, and potentially inhibiting, the activity of
KSP. In this embodiment, either component can be labeled. Thus, for
example, if the competitor is labeled, the presence of label in the
wash solution indicates displacement by the agent. Alternatively,
if the candidate agent is labeled, the presence of the label on the
support indicates displacement.
[0313] In an alternative embodiment, the candidate agent is added
first, with incubation and washing, followed by the competitor. The
absence of binding by the competitor may indicate the candidate
agent is bound to KSP with a higher affinity. Thus, if the
candidate agent is labeled, the presence of the label on the
support, coupled with a lack of competitor binding, may indicate
the candidate agent is capable of binding to KSP.
[0314] Inhibition is tested by screening for candidate agents
capable of inhibiting the activity of KSP comprising the steps of
combining a candidate agent with KSP, as above, and determining an
alteration in the biological activity of KSP. Thus, in this
embodiment, the candidate agent should both bind to KSP (although
this may not be necessary), and alter its biological or biochemical
activity as defined herein. The methods include both in vitro
screening methods and in vivo screening of cells for alterations in
cell cycle distribution, cell viability, or for the presence,
morpohology, activity, distribution, or amount of mitotic spindles,
as are generally outlined above.
[0315] Alternatively, differential screening may be used to
identify drug candidates that bind to the native KSP, but cannot
bind to modified KSP.
[0316] Positive controls and negative controls may be used in the
assays. Suitably all control and test samples are performed in at
least triplicate to obtain statistically significant results.
Incubation of all samples is for a time sufficient for the binding
of the agent to the protein. Following incubation, all samples are
washed free of non-specifically bound material and the amount of
bound, generally labeled agent determined. For example, where a
radiolabel is employed, the samples may be counted in a
scintillation counter to determine the amount of bound
compound.
[0317] A variety of other reagents may be included in the screening
assays. These include reagents like salts, neutral proteins, e.g.,
albumin, detergents, etc which may be used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Also reagents that otherwise improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors,
anti-microbial agents, etc., may be used. The mixture of components
may be added in any order that provides for the requisite
binding.
Administration
[0318] Accordingly, the compounds of the invention are administered
to cells. By "administered" herein is meant administration of a
therapeutically effective dose of a compound of the invention to a
cell either in cell culture or in a patient. By "therapeutically
effective dose" herein is meant a dose that produces the effects
for which it is administered. The exact dose will depend on the
purpose of the treatment, and will be ascertainable by one skilled
in the art using known techniques. As is known in the art,
adjustments for systemic versus localized delivery, age, body
weight, general health, sex, diet, time of administration, drug
interaction and the severity of the condition may be necessary, and
will be ascertainable with routine experimentation by those skilled
in the art. By "cells" herein is meant any cell in which mitosis or
meiosis can be altered.
[0319] A "patient" for the purposes of the present invention
includes both humans and other animals, particularly mammals, and
other organisms. Thus the methods are applicable to both human
therapy and veterinary applications. In a particular embodiment the
patient is a mammal, and more particularly, the patient is
human.
[0320] Compounds of the invention having the desired
pharmacological activity may be administered, generally as a
pharmaceutically acceptable composition comprising an
pharmaceutical excipient, to a patient, as described herein.
Depending upon the manner of introduction, the compounds may be
formulated in a variety of ways as discussed below. The
concentration of therapeutically active compound in the formulation
may vary from about 0.1-100 wt. %.
[0321] The agents may be administered alone or in combination with
other treatments, i.e., radiation, or other chemotherapeutic agents
such as the taxane class of agents that appear to act on
microtubule formation or the camptothecin class of topoisomerase I
inhibitors. When used, other chemotherapeutic agents may be
administered before, concurrently, or after administration of a
compound of the present invention. In one aspect of the invention,
a compound of the present invention is co-administered with one or
more other chemotherapeutic agents. By "co-administer" it is meant
that the present compounds are administered to a patient such that
the present compounds as well as the co-administered compound may
be found in the patient's bloodstream at the same time, regardless
when the compounds are actually administered including
simultaneously.
[0322] The administration of the compounds and compositions of the
present invention can be done in a variety of ways, including, but
not limited to, orally, subcutaneously, intravenously,
intranasally, transdermally, intraperitoneally, intramuscularly,
intrapulmonary, vaginally, rectally, or intraocularly. In some
instances, for example, in the treatment of wounds and
inflammation, the compound or composition may be directly applied
as a solution or spray.
[0323] Pharmaceutical dosage forms include a compound of formula I
or a pharmaceutically acceptable salt, solvate, or solvate of a
salt thereof, and one or more pharmaceutical excipients. As is
known in the art, pharmaceutical excipients are secondary
ingredients which function to enable or enhance the delivery of a
drug or medicine in a variety of dosage forms (e.g.: oral forms
such as tablets, capsules, and liquids; topical forms such as
dermal, opthalmic, and otic forms; suppositories; injectables;
respiratory forms and the like). Pharmaceutical excipients include
inert or inactive ingredients, synergists or chemicals that
substantively contribute to the medicinal effects of the active
ingredient. For example, pharmaceutical excipients may function to
improve flow characteristics, product uniformity, stability, taste,
or appearance, to ease handling and administration of dose, for
convenience of use, or to control bioavailability. While
pharmaceutical excipients are commonly described as being inert or
inactive, it is appreciated in the art that there is a relationship
between the properties of the pharmaceutical excipients and the
dosage forms containing them.
[0324] Pharmaceutical excipients suitable for use as carriers or
diluents are well known in the art, and may be used in a variety of
formulations. See, e.g., Remington's Pharmaceutical Sciences, 18th
Edition, A. R. Gennaro, Editor, Mack Publishing Company (1990);
Remington: The Science and Practice of Pharmacy, 20th Edition, A.
R. Gennaro, Editor, Lippincott Williams & Wilkins (2000);
Handbook of Pharmaceutical Excipients, 3rd Edition, A. H. Kibbe,
Editor, American Pharmaceutical Association, and Pharmaceutical
Press (2000); and Handbook of Pharmaceutical Additives, compiled by
Michael and Irene Ash, Gower (1995), each of which is incorporated
herein by reference for all purposes.
[0325] Oral solid dosage forms such as tablets will typically
comprise one or more pharmaceutical excipients, which may for
example help impart satisfactory processing and compression
characteristics, or provide additional desirable physical
characteristics to the tablet. Such pharmaceutical excipients may
be selected from diluents, binders, glidants lubricants,
disintegrants, colors, flavors, sweetening agents, polymers, waxes
or other solubility-retarding materials.
[0326] Compositions for intravenous administration will generally
comprise intravenous fluids, i.e., sterile solutions of simple
chemicals such as sugars, amino acids or electrolytes, which can be
easily carried by the circulatory system and assimilated. Such
fluids are prepared with water for injection USP.
[0327] Fluids used commonly for intravenous (IV) use are disclosed
in Remington, the Science and Practice of Pharmacy [full citation
previously provided], and include:
[0328] alcohol (e.g., in dextrose and water ("D/W") [e.g., 5%
dextrose] or dextrose and water [e.g., 5% dextrose] in normal
saline solution ("NSS"); e.g. 5% alcohol);
[0329] synthetic amino acid such as Aminosyn, FreAmine, Travasol,
e.g., 3.5 or 7; 8.5; 3.5, 5.5 or 8.5% respectively;
[0330] ammonium chloride e.g., 2.14%;
[0331] dextran 40, in NSS e.g., 10% or in D5/W e.g., 10%;
[0332] dextran 70, in NSS e.g., 6% or in D5/W e.g., 6%;
[0333] dextrose (glucose, D5/W) e.g., 2.5-50%;
[0334] dextrose and sodium chloride e.g., 5-20% dextrose and
0.22-0.9% NaCl;
[0335] lactated Ringer's (Hartmann's) e.g., NaCl 0.6%, KCl 0.03%,
CaCl.sub.2 0.02%;
[0336] lactate 0.3%;
[0337] mannitol e.g., 5%, optionally in combination with dextrose
e.g., 10% or NaCl e.g., 15 or 20%;
[0338] multiple electrolyte solutions with varying combinations of
electrolytes, dextrose, fructose, invert sugar Ringer's e.g., NaCl
0.86%, KCl 0.03%, CaCl.sub.2 0.033%;
[0339] sodium bicarbonate e.g., 5%;
[0340] sodium chloride e.g., 0.45, 0.9, 3, or 5%;
[0341] sodium lactate e.g., 1/6 M, and
[0342] sterile water for injection
The pH of such fluids may vary, and will typically be from 3.5 to 8
such in the art.
[0343] The following examples serve to more ftilly describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All publications, including
but not limited to patents and patent applications, cited in this
specification are herein incorporated by reference as if each
individual publication were specifically and individually indicated
to be incorporated by reference herein as though fully set
forth.
EXAMPLES
Example 1
[0344] Synthesis of Compounds ##STR20## ##STR21##
[0345] A solution of 4-chloro-2-iodobenzoic acid 1 (25 g, 88.7
mmol), SOCl.sub.2 (100 mL), and DMF (few drops) was warmed gently
with a heat gun until the mixture became homogeneous (15 mins). The
solution was maintained at 23.degree. C. for an additional 30 mins,
then the solution was concentrated. MeOH (200 mL) was added to the
crude residue and the solution was maintained at 23.degree. C. for
30 mins. The solution was concentrated and the crude residue was
then dissolved in 10:1 hexanes:EtOAc and passed through a plug of
silica gel. The eluent was concentrated to provide 26.2 g (100%) of
ester 2 as a colorless oil, which solidified upon standing under
high vacuum (0.1 Torr).
[0346] A mixture of ester 2 (2.35 g, 7.94 mmol), acetylene 31 (1.72
g, 8.7 mml), Pd(PPh.sub.3)Cl.sub.2 (140 mg, 0.20 mmol), Cul (19 mg,
0.1 mmol), and TEA (35 mL) was maintained at 50.degree. C. for 1 h.
The reaction mixture was diluted with EtOAc (200 mL), washed with
water (100 mL), then brine (100 mL). The organic layer was dried
(MgSO.sub.4) and concentrated. The crude residue was purified by
flash column chromatography (10:1 hexanes:EtOAc) to provide 2.88 g
(100%) of 4. 1 Fehrentz, J.-A.; Castro, B. Synthesis 1983, 676.
Goel. O.; Krolls, U.; Steir, M.; Kesten, S. Org. Syn. 1988, 67,
69.
[0347] Ester 4 (2.88 g, 7.94.mmol), KOH (930 mg, 16.5 mmol), MeOH
(15 mL), THF (15 mL), and H.sub.2O (15 mL) was maintained at
50.degree. C. for exactly 30 mins. The reaction was diluted with
EtOAc (100 mL) and washed with 1N HCl (20 mL) and brine (20 mL).
The organic layer was dried (MgSO.sub.4) and concentrated to
provide 2.76 g of acid 5 (99%), which was used without further
purification.
[0348] Pd(MeCN).sub.2Cl.sub.2 (98mg, 0.39 mmol) was added to a
solution of the crude acid 5 (2.76 g, 7.87 mmol), TEA (3.3 mL) and
THF (54 mL). The solution was maintained at 50.degree. C. for 30
mins. The reaction solution was diluted with hexanes (150 mL) and
passed through a plug of silica gel. The plus was rinsed with (5:1
hexanes:EtOAc). The filtrate was concentrated. Hexanes (50 mL) was
added to this crude residue and pure 6 precipitated out of
solution. This precipitated material was filtered and used without
further purification. The filtrate was then concentrated and the
remaining residue dried onto silica gel (CH.sub.2Cl.sub.2) and
purified by flash column chromatography (10:1 hexanes:EtOAc; 5:1
hexanes:EtOAc; 3:1 hexanes:EtOAc) to provide a combined 2.22 g of 6
(77%, two steps).
[0349] A solution of isochromenone 6 (2.1 g, 6.0 mmol), BnNH.sub.2
(1.96 mL, 17.9 mmol), and PhMe (3.0 mL) was maintained at
140.degree. C. for 1.5 h. The reaction mixture was cooled to
23.degree. C., diluted with EtOAc (50 mL), washed with 1 N HCl (20
mL) and brine (20 mL). The organic layer was dried (MgSO.sub.4) and
concentrated. The resulting crude residue was used without further
purification. A solution of the crude residue, 5% HCl (3.4 mL) and
MeOH (17 ml) was maintained at 50.degree. C. for 1.5 h. The product
7 begins to precipitate from the reaction solution after 5 mins as
a white solid. After 30 mins, an additional amount of MeOH (5 mL)
and 5% HCl (1 mL) was added and the reaction mixture was maintained
for an additional 1 h. The reaction mixture was diluted with EtOAc
(100 mL), washed with 1 N NaOH (50 mL) and then brine (20 mL). The
organic layer was dried (MgSO.sub.4) and concentrated. The residue
was purified by flash column chromatography (5:1 hexanes:EtOAc) to
provide 1.8 g of racemic 7 (68%, two steps).
[0350] A solution of isoquinolone 7 (2.50 g, 5.68 mmol) and
TFA:H.sub.2O (97.5:2.5; 25 mL) was maintained at 23.degree. C. for
1 h. The solution was concentrated. The crude residue was diluted
with EtOAc (75 mL), washed with 1 N NaOH (20 mL), and brine (20
mL). The organic layer was dried (MgSO.sub.4) and concentrated to
give 1.83 g of amine 9 (95%) as a white solid.
[0351] Isoquinolone 8 (131 mg, 0.34 mmol), aldehyde 9 (115 mg, 0.56
mmol), Na(OAc).sub.3BH (291 mg, 1.38 mmol), and CH.sub.2Cl.sub.2
(1.1 mL) was maintained at 23.degree. C. for 3 h. The reaction
mixture was diluted with EtOAc (20 mL) and washed with 1 N NaOH (5
mL) and brine (5 mL). The organic layer was dried (MgSO.sub.4),
filtered, and concentrated. The resulting residue was purified by
flash column chromatography (3:1 hexanes:EtOAc) to yield 150 mg
(85%) of 10.
[0352] A solution of isoquinolone 10 (143 mg, 0.28 mmol), POCl3
(0.45 mL, 4.8 mmol), and PhMe (14 mL) was heated to 110.degree. C.
After 6 h, the reaction mixture was diluted with EtOAc (40 mL) and
washed with 1 N NaOH (20 mL) and brine (10 mL). The organic layer
was dried (MgSO.sub.4), filtered, and concentrated. The resulting
residue was purified by flash column chromatography (20:1
CHCl.sub.3:MeOH) to yield 99 mg (70%) of 11 as a white solid.
Example 2
[0353] Synthesis of Compounds ##STR22##
[0354] Isoquinolone 8 (515 mg, 1.47 mmol), aldehyde 12 (255 mg,
1.47 mmol), NaCN(OAc).sub.3BH (420 mg, 1.98 mmol), and
CH.sub.2Cl.sub.2 (4.1 mL) was maintained at 23.degree. C. for 2 h.
An additional portion of 12 (225 mg, 1.30 mmol) in CH.sub.2Cl.sub.2
(0.6 mL) was then added. After an additional 3 h, the reaction
mixture was diluted with EtOAc (20 mL) and washed with 1 N NaOH (5
mL) and brine (5 mL). The organic layer was dried (MgSO.sub.4),
filtered, and concentrated. The resulting residue was purified by
flash column chromatography (3:1 hexanes:EtOAc; 1:1 hexanes:EtOAc)
to yield 630 mg (86%) of 13.
[0355] To a solution of isoquinolone 13 (85 mg, 0.17 mmol),
diisoproylethylamine (DIEA, 0.12 mL, 0.68 mmol), and
CH.sub.2Cl.sub.2 (0.6 mL) at 23.degree. C. was added p-toluoyl
chloride (45 .mu.L, 34 mmol). After 4 h, the reaction mixture was
diluted with EtOAc (20 mL) and washed with saturated aqueous
NaHCO.sub.3 (5 mL) and brine (5 mL). The organic layer was dried
(MgSO.sub.4), filtered, and concentrated. The resulting residue was
purified by flash column chromatography (3:1 hexanes:EtOAc) to
yield 83 mg (80%) of 14.
[0356] Isoquinolone 14 (80 mg, 0.13 mmol) and TFA:H.sub.2O
(97.5:2.5, 2 mL) was maintained at 23.degree. C. for 1 h. The
reaction mixture was concentrated. The residue was dissolved in
EtOAc (20 mL) and washed with 1 N NaOH (5 mL) and brine (5 mL). The
organic layer was dried (MgSO.sub.4), filtered, and concentrated to
provide 65 mg (98%) of 15 as a white solid which was deemed >95%
pure by .sup.1H NMR and LCMS analysis.
Example 3
[0357] Using the methods of the invention as exemplified in
Examples 1 and 2 above, the following compounds were prepared:
TABLE-US-00001 LRMS Structure (MH) m/z ##STR23## 516.2 ##STR24##
470.2 ##STR25## 512.2 ##STR26## 575.8 ##STR27## 528.0 ##STR28##
464.0 ##STR29## 530.0 ##STR30## 516.2 ##STR31## 566.2 ##STR32##
512.2 ##STR33## 565.8 ##STR34## 558.2
Example 4
Monopolar Spindle Formation Following Application of an
Isoquinolone KSP Inhibitor
[0358] Human tumor cell lines Skov-3 (ovarian) are plated in
96-well plates at densities of 4,000 cells per well, allowed to
adhere for 24 hours, and treated with various concentrations of the
isoquinolone compounds for 24 hours. Cells are fixed in 4%
formaldehyde and stained with antitubulin antibodies (subsequently
recognized using fluorescently-labeled secondary antibody) and
Hoechst dye (which stains DNA).
[0359] Visual inspection reveals that the compounds cause cell
cycle arrest in the prometaphase stage of mitosis. DNA is condensed
and spindle formation is initiated, but arrested cells uniformly
display monopolar spindles, indicating that there is an inhibition
of spindle pole body separation. Microinjection of anti-KSP
antibodies also causes mitotic arrest with arrested cells
displaying monopolar spindles.
Example 5
Inhibition of Cellular Proliferation in Tumor Cell Lines Treated
with KSP Inhibitors.
[0360] Cells are plated in 96-well plates at densities from
1000-2500 cells/well of a 96-well plate and allowed to adhere/grow
for 24 hours. They are then treated with various concentrations of
drug for 48 hours. The time at which compounds are added is
considered T.sub.0. A tetrazolium-based assay using the reagent
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium (MTS) (1.S>U.S. Pat. No. 5,185,450) (see Promega
product catalog #G3580, CellTiter 96.RTM. AQ.sub.ueous One Solution
Cell Proliferation Assay) is used to determine the number of viable
cells at T.sub.0 and the number of cells remaining after 48 hours
compound exposure. The number of cells remaining after 48 hours is
compared to the number of viable cells at the time of drug
addition, allowing for calculation of growth inhibition.
[0361] The growth over 48 hours of cells in control wells that have
been treated with vehicle only (0.25% DMSO) is considered 100%
growth and the growth of cells in wells with compounds is compared
to this. KSP inhibitors inhibit cell proliferation in human ovarian
tumor cell lines (SKOV-3).
[0362] A Gi.sub.50 is calculated by plotting the concentration of
compound in .mu.M vs the percentage of cell growth of cell growth
in treated wells. The Gi.sub.50 calculated for the compounds is the
estimated concentration at which growth is inhibited by 50%
compared to control, i.e., the concentration at which:
100.times.[(Treated.sub.48-T.sub.0)/(Control.sub.48-T.sub.0)]=50.
[0363] All concentrations of compounds are tested in duplicate and
controls are averaged over 12 wells. A very similar 96-well plate
layout and GI.sub.50 calculation scheme is used by the National
Cancer Institute (see Monks, et al., J. Natl. Cancer Inst. 83
:757-766 (1991)). However, the method by which the National Cancer
Institute quantitates cell number does not use MTS, but instead
employs alternative methods.
Example 6
Calculation of IC.sub.50:
[0364] Measurement of a compound's IC.sub.50 for KSP activity uses
an ATPase assay. The following solutions are used: Solution 1
consists of 3 mM phosphoenolpyruvate potassium salt (Sigma P-7127),
2 mM ATP (Sigma A-3377), 1 mM IDTT (Sigma D-9779), 5 .mu.M
paclitaxel (Sigma T-7402), 10 ppm antifoam 289 (Sigma A-8436), 25
mM Pipes/KOH pH 6.8 (Sigma P6757), 2 mM MgCl2 (VWR JT400301), and 1
mM EGTA (Sigma E3889). Solution 2 consists of 1 mM NADH (Sigma
N8129), 0.2 mg/ml BSA (Sigma A7906), pyruvate kinase 7U/ml,
L-lactate dehydrogenase 10 U/ml (Sigma P0294), 100 nM KSP motor
domain, 50 .mu.g/ml microtubules, 1 mM DTT (Sigma D9779), 5 .mu.M
paclitaxel (Sigma T-7402), 10 ppm antifoam 289 (Sigma A-8436), 25
mM Pipes/KOH pH 6.8 (Sigma P6757), 2 mM MgCl2 (VWR JT4003-01), and
1 mM EGTA (Sigma E3889). Serial dilutions (8-12 two-fold dilutions)
of the compounds are made in a 96-well microtiter plate (Corning
Costar 3695) using Solution 1. Following serial dilution each well
has 50 .mu.l of Solution 1. The reaction is started by adding 50
.mu.l of Solution 2 to each well. This may be done with a
multichannel pipettor either manually or with automated liquid
handling devices. The microtiter plate is then transferred to a
microplate absorbance reader and multiple absorbance readings at
340 nm are taken for each well in a kinetic mode. The observed rate
of change, which is proportional to the ATPase rate, is then
plotted as a function of the compound concentration. For a standard
IC.sub.50 determination the data acquired is fit by the following
four parameter equation using a nonlinear fitting program (e.g.,
Grafit 4): y = Range 1 + ( x IC 50 ) s + Background ##EQU2## where
y is the observed rate and x the compound concentration.
* * * * *
References